Formulations for small intestinal delivery of rsv and norovirus antigens

ABSTRACT

Provided herein are compositions and methods for generating an immunogenic response in humans. Further provided are methods for designing such compositions, e.g., for vaccines.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims is a continuation of application Ser. No.15/580,651 filed Dec. 7, 2017, which is a 371 national stage applicationof PCT Application No. PCT/US2016/036461, filed Jun. 8, 2016, whichclaims benefit of U.S. Provisional Patent Application No. 62/175,081,filed Jun. 12, 2015, the disclosures of which are each incorporated byreference for all purposes.

REFERENCE TO SUBMISSION OF A SEQUENCE LISTING AS AN XML FILE

The Sequence Listing written in file 090402-001620US-1325836.xml,created on Aug. 1, 2022, 53,484 bytes, machine format IBM-PC, MS-Windowsoperating system, is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Vaccines are an important means for preventing and/or treating a numberof diseases and disorders (e.g., viral infection, bacterial infection,and cancer). Vaccinization is typically carried out using injection,which reduces participation due to inconvenience of traveling to avaccination site and aversion to injections. Furthermore, injection ofvaccines requires use of sterile kit, such as syringes and needles, anda skilled practitioner to administer.

For the influenza vaccine, large-scale yearly campaigns are conducted tocollect enough fertilized eggs to harvest and process sufficient virusto meet the needs of the market. Cell culture or plant derivedhemagglutinin (HA) may reduce the burden of egg acquisition andprocessing, but these approaches still require expensive sterile filland finish to produce individual syringe needles, that need to bedisposed of as a biohazard. During a pandemic, schools can be closed andsocial distancing mandated, yet mass influenza immunization typicallyrequires lining up subjects at health clinics for injections. Oralvaccines, for influenza or other pathogens, could be sent through themail thus avoiding most human to human contact. Further, tableting is arapid, sanitary process that does not require the expensive sterile filland finishing process that injected vaccines require.

Vaccines that can be delivered in a non-parenteral manner, e.g., orallyor mucosally, are described in U.S. Pat. No. 8,222,224.

BRIEF SUMMARY OF THE INVENTION

Provided herein are methods and compositions for more effectivevaccination of a subject (human or non-human) involving delivery of animmunogenic biological agent specifically to the ileum of the subject.The present disclosure thus provides more efficient and effectivevaccines, and demonstrates their effectiveness in humans.

Provided herein are immunogenic compositions for eliciting an immuneresponse in a subject comprising: an immunogenic biological agentencompassed by a delivery agent that directs delivery of the immunogenicbiological agent to the ileum of the subject. In some embodiments, thesubject is a human. In some embodiments, the subject is a non-humananimal, e.g., primate, mouse, rat, rabbit, horse, dog, cat, or poultry.In some embodiments, the immunogenic biological agent is selected froman immunogenic polypeptide (e.g., virus like particle, glycoprotein,phosphoprotein), carbohydrate, and lipid. In some embodiments, theimmunologenic biological agent is an adenoviral vector encoding theviral protein 1 of norovirus or the fusion protein (F) of Respiratorysyncytial virus (RSV). In some embodiments, the viral protein 1 ofnorovirus is SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, thefusion protein (F) of RSV is SEQ ID NO: 6. In some embodiments, theadenoviral vector comprises a nucleotide sequence of SEQ ID NO: 7.

In some embodiments, the immunogenic biological agent is an expressionvector encoding an immunogenic polypeptide. In some embodiments, theexpression vector is a viral vector (e.g., adenoviral, AAV, retroviral,or lentiviral). In some embodiments, the viral vector is attenuated orreplication incompetent. In some embodiments, the expression vectorcomprises a promoter (e.g., CMV, SV40 early or late, β-actin, etc.)operably linked to the sequence encoding the immunogenic polypeptide. Insome embodiments, the expression vector further encodes double stranded(dsRNA). In some embodiments, the dsRNA encoding sequence is operablylinked to a promoter, e.g., either the same promoter (using an InternalRibosomal Entry Site (IRES)) or a different promoter as the promoteroperably linked to the immunogenic polypeptide encoding sequence.

In some embodiments, the immunogenic composition further comprises atleast one adjuvant, e.g., a TLR3 agonist. In some embodiments, the TLR3agonist is dsRNA or a dsRNA mimetic.

In some embodiments, at least 50% of the immunogenic biological agent isdelivered (released) in the ileum, e.g., at least 60%, 70%, 75%, 80%,90%, 95%, or more of the immunogenic biological agent present in theadministered composition. In some embodiments, the enteric coating ormatrix begins to dissolve before the immunogenic composition reaches theileum, but retains at least 50% of the immunogenic biological agentuntil the immunogenic composition reaches the ileum. In someembodiments, the enteric coating retains the immunogenic biologicalagent through the stomach, duodenum, and jejunum, but releases theimmunogenic biological agent in the ileum.

In some embodiments the immunogenic biological agent is covered by anenteric coating. In some embodiments, the enteric coating disintegratesat pH≥5, e.g., 5.2, 5.5, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 7.0, 5.5-6.8, 5.8-6.8, etc. In some embodiments, the entericcoating is selected from the group consisting of methacrylic acid-ethylacrylate copolymer (e.g., 1:1), type A; methacrylic acid copolymer, typeC; a mixture of methacrylic copolymer types A and C; and Time Clock®. Insome embodiments, the enteric coating does not include cellulose acetatephthalate (CAP). In some embodiments, the enteric coating is of athickness that results in release of the immunogenic biological agent inthe ileum. In some embodiments, the enteric coating is methacrylic acidcopolymer-based with a coverage of 5.5-10 milligram per squarecentimeter. In some embodiments, the delivery agent is aradio-controlled capsule.

In some embodiments, the enteric coating comprises poly(methacrylicacid-co-methyl methacrylate) 1:1. In some embodiments, the entericcoating comprises Eudragit® L-100. In some embodiments, the entericcoating comprises Eudragit® L-100, triethyl citrate, and talc, e.g., 1,2, 3, 4 or 1-4 parts Eudragit® L-100, 1-2 parts triethyl citrate, and1-2 parts talc. In some embodiments, enteric coating comprises a mixtureof poly(methacrylic acid-co-methyl methacrylate) 1:1 andpoly(methacrylic acid-co-ethyl acrylate) 1:1. In some embodiments, theratio of poly(methacrylic acid-co-methyl methacrylate) 1:1 topoly(methacrylic acid-co-ethyl acrylate) 1:1 is 1:4 to 4:1, e.g., 1:3,1:2, 1:1, 2:1, 3:1. In some embodiments, the enteric coating comprises amixture of Eudragit® L-100 and Eudragit®L100-55. In some embodiments,the enteric coating comprises Eudragit® L-100 and Eudragit®L100-55,triethyl citrate, and talc, e.g., 1-4 parts Eudragit® L-100 andEudragit®L100-55, 1-2 parts triethyl citrate, and 1-2 parts talc. Insome embodiments, the enteric coating comprises poly(methacrylicacid-co-methyl methacrylate) 1:1 and poly(methacrylic acid-co-methylmethacrylate) 1:2. In some embodiments, the ratio of poly(methacrylicacid-co-methyl methacrylate) 1:1 to poly(methacrylic acid-co-methylacrylate) 1:2 is 1:2 to 2:1. In some embodiments, the enteric coatingcomprises a mixture of Eudragit® L-100 and Eudragit®S100. In someembodiments, the enteric coating comprises Eudragit® L-100 andEudragit®S100, triethyl citrate, and talc, e.g., 1-4 parts Eudragit®L-100 and Eudragit®S100, 1-2 parts triethyl citrate, and 1-2 parts talc.In some embodiments, the enteric coating comprises a mixture ofpoly(methacrylic acid-co-methyl methacrylate) 1:2 and poly(methacrylicacid-co-ethyl acrylate) 1:1. In some embodiments, the ratio ofpoly(methacrylic acid-co-methyl methacrylate) 1:2 and poly(methacrylicacid-co-ethyl acrylate) 1:1 is 1:4 to 4:1, e.g., 1:3, 1:2, 1:1, 2:1, or3:1. In some embodiments, the enteric coating comprises a mixture ofEudragit® L-100-55 and Eudragit®S100. In some embodiments, the entericcoating comprises Eudragit® L-100-55 and Eudragit®S100, triethylcitrate, and talc, e.g., 1-4 parts Eudragit® L-100-55 and Eudragit®S100,1-2 parts triethyl citrate, and 1-2 parts talc.

In some embodiments, the immunogenic composition is in the form of atablet or capsule, e.g., in the form of a compressed tablet covered byenteric coating. In some embodiments, the immunogenic composition isencapsulated in a polymeric capsule comprising gelatin,hydroxypropylmethylcellulose, starch, or pullulan. In some embodiments,the immunogenic composition is in the form of microparticles less than 2mm in diameter, e.g., each microparticle covered with enteric coating asdescribed herein.

Further provided is a method of delivering an immunogenic composition tothe ileum of a subject comprising orally administering the immunogeniccomposition as described above (i.e., an immunogenic biological agentencompassed by a delivery agent that directs delivery of the immunogenicbiological agent to the ileum, optionally including an adjuvant) to thesubject. In some embodiments, the subject is a human. In someembodiments, the subject is a non-human animal. In some embodiments, themethod results in an immune response in the subject that is at least 10%higher, e.g., at least 20%, 30%, 40%, 50%, 60%, 75%, 80%, 100% or more,than the immune response in a subject (either the same subject at adifferent time, or a different subject) receiving the same immunogeniccomposition not directed to the ileum. In some embodiments, the immuneresponse in the subject is at least 1.5-fold higher (e.g., 2-fold,2.5-fold, 5-fold, or more) than the immune response in a subject (eitherthe same subject at a different time, or a different subject) receivingthe same immunogenic composition not directed to the ileum. In someembodiments, the immune response is an increase in antibodies specificfor the immunogenic biological agent. In some embodiments, the immuneresponse is a cellular immune response, e.g., an increase in cytokinessuch as IFN-γ. In some embodiments, the immune response is immunization(e.g., the subject is resistant to infection by the virus, bacteria,etc. from which the immunogenic biological agent was derived).

Further provided are methods of eliciting an increased immune responsein a subject comprising orally administering the immunogenic compositionas described above (i.e., an immunogenic biological agent encompassed bya delivery agent that directs delivery of the immunogenic biologicalagent to the ileum, optionally including an adjuvant) to the subject,e.g., human subject. In some embodiments, the immune response isincreased by at least 10%, e.g., at least 20%, 30%, 40%, 50%, 60%, 75%,80%, 100% or more, compared to the immune response in a subject (eitherthe same subject at a different time, or a different subject) receivingthe same immunogenic composition not directed to the ileum. In someembodiments, the immune response in the subject is increased at least1.5-fold (e.g., 2-fold, 2.5-fold, 5-fold, or more) compared the immuneresponse in a subject (either the same subject at a different time, or adifferent subject) receiving the same immunogenic composition notdirected to the ileum. In some embodiments, the immune response is anincrease in antibodies specific for the immunogenic biological agent. Insome embodiments, the immune response is a cellular immune response,e.g., an increase in cytokines such as IFN-γ. In some embodiments, theimmune response is immunization (e.g., the subject is resistant toinfection by the virus, bacteria, etc. from which the immunogenicbiological agent was derived).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Antibody Secreting Cells (ASCs) specific for HA were measuredin the peripheral blood 7 days after subjects were given aradio-controlled capsule containing rAd-HA-dsRNA. Subjects wererandomized to have the vaccine released in either the ileum or jejunum.(N=12 per group). Results show that 12 of 12 subjects with vaccinedelivered to the ileum were able to generate antibody secreting B cellsthat recognize HA, whereas only 9 of 12 subjects given the vaccine tothe jejunum were able to generate antigen specific B cells. The averagenumber of IgA and IgG ASCs was significantly higher for the ileum thanthe jejunum.

FIG. 2 . T cell response to rAd-HA-dsRNA was determined by detectingIFN-γ levels 7 days post-administration. All of the individuals in theileum-delivery group showed higher levels of IFN-γ, compared to 75% ofthe jejunum-delivery group. The average IFN-γ level was alsosignificantly higher in the ileum-delivery group.

FIG. 3 . Microneutralizing antibody (MN) responses to influenzaA/CA/07/2009 were measured at day 0 and day 28 after immunization. Thefold increase in MN titers was plotted for individual subjects that hadan initial MN titer less than or equal to 40. Results showed that ileumdelivery resulted in a high proportion of subjects (9 of 10) withincreased MN titers following immunization compared to jejunum delivery(6 of 10).

FIG. 4A-4B. Tablets were made using microcrystalline cellulose andstarch, with 10% barium sulfate as a radiopaque material. These tabletswere enteric coated with Eudragit L100® and given to female cynomolgusmacaques by oral gastric tube. X-rays were taken over time postadministration. FIG. 4A. Tablet in the stomach with the arrow pointingtoward the tablet. FIG. 4B. One hour later, the tablet can be seen inthe intestine, the white spot to the left of the spinal column with anarrow point toward it. It dissolved in the intestine within the next twohours, and cannot be seen.

FIG. 5 . The numbers of ASCs are reported on days 7 and 35, 7 days aftereach immunization. Background ASCs at days 0 and 28 were miniscule, andnot plotted. Average responses for day 7 are shown for each treatedgroup with a horizontal line.

FIG. 6 . Fold increase in MN titers for individual subjects. The darkshaded columns indicate where the titers rose between days 28 and 56,whereas the light shaded columns shows the response after the initialimmunization. A line was drawn at two fold increases in MN to show whichsubjects had a detectable neutralizing antibody response. No subject inthe placebo group responded, whereas 3 subjects in the low dose and 7subjects in the high dose group had a 2 fold or greater neutralizingantibody response to influenza after immunization. Placebo N=10, LowDose and High Dose N=11.

FIG. 7A-7D. Antibody responses following a single oral immunization.FIG. 7A. Hemagglutination Inhibition (“HAI”) antibody titers pre andpost immunization (days 0 and 28 respectively) are shown for individualsubjects. FIG. 7B. HAI Geometric Mean Titers (GMT) vs Time. HAI titerswere measured at 0, 1, and 6 months post immunization to evaluate thedurability of the antibody response. FIG. 7C. MN titers, pre and postimmunization are shown for individual subjects. FIG. 7D. ASC responsesfollowing immunization. The numbers of IgG and IgA ASCs are reported(per 10⁶ PMBCs) 7 days after immunization.

FIG. 8 . Serum ELISA IgG Titers Versus Dose of VXA-G1.1-NN in mice. Micewere immunized with VXA-G1.1-NN of 1×108, 5×108 and 1×109. VXA-G1.1-NNwas delivered orally by gavage on days 0 and 28. The serum IgG responsesagainst Norwalk VP1 were measure by ELISA at week 8. N=6. Each iconrepresents an individual mouse. The top of the bar for each studyindicates the GMT. As the dose was increased from 1×108 to 5×108 to1×109, the serum VP1 IgG titer showed a dose-dependent increase from2×103 to 1×104 to 5×105.

FIG. 9 . Fecal SIgA ELISA Titers Versus Dose of VXA-G1.1-NN in mice.Mice were immunized with VXA-G1.1-NN of 1×108, 5×108 and 1×109.VXA-G1.1-NN was delivered orally by gavage on days 0 and 28. The fecalSIgA responses against Norwalk VP1 were measure by ELISA at week 8. Eachstudy has a total of 6 mice. Each icon represents an individual mouse.The top of the bar for each study indicates the GMT. As the dose wasincreased from 1×108 to 5×108 to 1×109, the fecal VP1 sIgA titer showeda dose-dependent increase from 1×103 to 2×103 to 3×104.

FIG. 10 . Serum ELISA IgG Titers of VXA-G1.1-NN Versus VP1 protein inmice. Mice were immunized with VXA-G1.1-NN of 1×108 orally or Norwalkvirus VP1 protein (1 ug) intramuscularly on days 0 and 28. The serum IgGresponses against Norwalk VP1 were measured by ELISA at weeks 4 and 8.Each study has a total of 6 mice. Each icon represents an individualmouse. The top of the bar for each study indicates the geomean titer.Oral vaccine generated slightly higher serum IgG titer values than thei.m. protein vaccine.

FIG. 11 . Fecal SIgA ELISA Titers Versus Dose of VXA-G1.1-NN in mice.Mice were immunized with VXA-G1.1-NN of 1×108 orally or Norwalk virusVP1 protein (1 ug) intramuscularly on days 0 and 28. The fecal IgAresponses against Norwalk VP1 were measure by ELISA at weeks 4 and 8.Each study has a total of 6 mice. Each icon represents an individualmouse. The top of the bar for each study indicates the geomean titer.The oral vaccine generated a dramatically higher fecal IgA immuneresponse than the i.m. protein vaccine.

FIG. 12 . Oral immunization of VXA-G1.1-NN compared to VP1 proteinimmunization for Serum ELISA Titers. Mice were immunized withVXA-G1.1-NN of 1×108 orally or Norwalk virus VP1 protein (1 ug) in thepresence of adjuvant, aluminium hydroxide intramuscularly on days 0 and28. The serum IgG responses against Norwalk VP1 were measure by ELISA atweeks 4 and 8. Each study has a total of 6 mice. Each icon represents anindividual mouse. The top of the bar for each study indicates thegeomean titer. Intramuscular injection with the VP1 protein togetherwith alum generated much higher serum titer.

FIG. 13 . Oral immunization of VXA-G1.1-NN compared to VP1 proteinimmunization for Fecal SIgA ELISA Titers Mice were immunized withVXA-G1.1-NN of 1×108 orally or Norwalk virus VP1 protein (1 ug) in thepresence of adjuvant, aluminium hydroxide intramuscularly on days 0 and28. The fecal IgA responses against Norwalk VP1 were measure by ELISA atweeks 4 and 8. Each study has a total of 6 mice. Each icon represents anindividual mouse. The top of the bar for each study indicates thegeomean titer. Even in the presence of adjuvant, aluminium hydroxide,the oral vaccine generated a higher fecal SIgA immune response than thei.m. protein vaccine.

FIG. 14A-14B. Serum and Fecal IgA Titers following Oral Immunization ofVXA-G2.4-NS in mice. Mice were immunized with VXA-G2.4-NS of 1×108orally on days 0 and 28. For comparison, oral delivered groups fromstudy#3 were presented again. The serum IgG responses against Sydney VP1were measure by ELISA at weeks 4 and 8 FIG. 14A. The fecal SIgA responseagainst the Sydney VP1 was measured by ELISA at week 8 FIG. 14B. At 4weeks, the Sydney strain vaccine generated better IgG titer values thanthe Norwalk vaccine. In addition, even at 4 weeks the titer values fromthe Sydney strain were slightly higher than the Norwalk values at 8weeks (A). The Sydney vaccine generated slightly higher fecal VP1 IgAtiter values than the Norwalk vaccine.

FIG. 15A-15B. Serum and Fecal IgA Titers following Oral Immunization ofVXA-G2.4-NS in ferrets. Ferrets were endoscopically administeredVXA-G2.4-NS on days 0 and 2 (group1) or days 0 and 28 (group2). Ferretsin group3 were intramuscularly administered the recombinant VP1 proteinfrom the Sydney strain norovirus on days 0 and 28. Whereas group1generated higher IgG titer values than group2, group2 generated higherSIgA titer values than group1. Group3 failed to generate fecal SIgAresponse although serum IgG responses were generated.

FIG. 16 . Serum IgG following Oral Immunization of VXA-G2.4-NS in Nonhuman primates (NHP, Cynomolgous macaque). NHPs were endoscopicallyadministered VXA-G2.4-NS on days 0 and 56 (the “Noro” group). Serum IgGtiters were measured by ELISA at day 0, 7, 28, 56, and 72.

FIG. 17 . Anti-RSVF antibody titers in cotton rats at 4weeks postvaccination with Ad-RSVF vaccine vectors or control formalin inactivatedRSV (FIRSV) or live wild-type RSVA2. Titers were determined using ananti-RSVF IgG ELISA.

FIG. 18 . Palivizumab Competition using sera from cotton rats vaccinatedwith Ad-RSVF vaccine. ELISA plates were coated with an RSVA2 lysate at 1ug/mL. Biotinylated Palivizumab (10 ng/mL) was mixed with serial 2-folddilutions of control and test sera. Naïve sera was used to determine100% Palivizumab binding. Unlabelled Palivizumab was used as positivecontrol. HRP-Strepavidin with TMB substrate was used to detectbiotinylated Palivizumab. Inhibition was scored relative to 100%biotinyaled Paliziumab binding. The maximum dilution that gave 50% orgreater neutralization activity was assigned as the competition titer.

FIG. 19 . Enrollment criteria for the study of “High Titer NeutralizingAntibodies to Influenza Following Oral Tablet Immunization: ARandomized, Placebo-controlled Trial.” The major exclusion criteria are:

-   -   positive for H1 influenza by HAI;    -   has had an influenza vaccine in the past 2 years;    -   current history of chronic alcohol consumption and/or illicit        and/or recreational drug use;    -   history of any confirmed or suspected immunodeficient or        immunosuppressive condition;    -   positive serology for HIV, HCV, or HBV;    -   previous serious reactions to vaccination such as anaphylaxis,        respiratory problems, hives, or abdominal pain;    -   history of irritable bowel disease or other inflammatory        digestive or gastrointestinal conditions that could affect the        intended distribution of the vaccine targeting the mucosa of the        small intestine;    -   use of proton pump inhibitors (Nexium, Prilosec); and    -   stool sample with occult blood at baseline exam.

FIGS. 20A-20D. Antibody responses following a single oral immunizationof a recombinant Ad serotype 5 (rAd5) based oral vaccine against H1seasonal influenza. FIG. 20A shows HAI antibody titers pre and postimmunization (days 0 and 28 respective) are shown for individualsubjects. FIG. 20B shows HAI GMT v. time. HAI titers were measured at 0,1, and 6 months post immunization to evaluate the durability of theantivody response. FIG. 20C shows MN titers pre and post immunizationfor individual subjects. FIG. 20D shows ASC response followingimmunization. The numbers of IgG and IgA ASCs are reported (per 1e6PMBCs) 7 days after immunization.

FIGS. 21A-21B. Anti-Ad5 immunity and effects on neutralizing antibodyresponses. FIG. 21A shows Fold change in MN versus starting anti-Ad5titer for vaccine treated subjects. Subjects were not pre-screened foranti-Ad5 titers, but retrospectively measured. FIG. 21B shows foldchange in HAI responses that were plotted for either Ad5 titer positiveor negative, analogous as performed in FIG. 2 . No trend was observedfor Ad5 starting titers on the ability to elicit a neutralizing antibodyresponse (by MN or HAI) to influenza virus.

FIG. 22 shows mice elicit robust antibody titers against RSV when givenVXA-RSV-f. Study No. WCB254: Balb/c mice were immunized with VXA-RSV-fat 0 and 3 weeks using three different routes of delivery. ELISA IgGantibody titers were measured at week 7. All routes of deliverygenerated significant immune responses against RSV; however, i.n. andi.m. were more effective at producing higher titers than oral (p=0.04,or 0.02 by Mann-Whitney). N=6 per group. As negative controls, mousesera from animals given a norovirus vaccine were used.

FIGS. 23A-23C shows immunization of cotton rats with VXA-RSV-f vaccineinduces antibody responses to RSV. (Experiment XV-95). FIG. 23A showsfemale cotton rats were immunized on week 0 and 4, and antibody titersagainst RSV-f were measured on week 8. VXA-RSV-f immunization wassignificantly better than using RSV2 virus or FI-RSV for inducing IgGELISA titers to RSV. (p<0.0022 by Mann-Whitney) FIG. 23B showspalivizumab competitive ELISA titers were evaluated using pooled serumsamples from each of the previously described groups. FI-RSV vaccine wasnot able to induce epitope specific antibodies that compete for thebinding of palivizumab, but the VXA-RSV-f vaccine and RSV2 treatedgroups were able to induce these titers. FIG. 23C shows the VXA-RSV-fvaccine induced neutralizing antibody responses to RSV followingimmunization of cotton rats. VXA-RSV-f and RSV2 groups were superior tothe FI-RSV, and the no vaccine controls (no infection and buffer groups)at inducing neutralizing titers against RSV. N=6 per group, except the“no infection” control (N=3).

FIGS. 24A-24C show oral immunization induces potent antibodies to RSV incotton rats. (Experiment XV-112). In FIG. 24A, female cotton rats wereimmunized with VXA-RSV-f on week 0 and 4, and the total IgG antibodyELISA titers were measured on weeks 4 and 8. Dose dependent antibodyresponses were observed with 1×10⁹ and 1×10¹⁰ trending better than the1×10⁸ IU dose group. In FIG. 24B, Palivizumab competitive ELISA titerswere evaluated for the same groups as described before, but at week 8.Higher vaccine doses trended with higher Palivizumab competitiveantibody titers, but the results were not significantly different. FIG.24C shows oral immunization of VXA-RSV-f also induced dose dependentneutralizing antibody titers to RSV, with the 1×1010 group performingsignificantly better than the 1×108 group (p=0.018 by Mann-Whitney). N=8per oral group, N=6 for buffer control group.

FIGS. 25A-25C shows VXA-RSV-f protects against RSV replication andadaptive immune enhancements of RSV disease. (Experiment XV-95) FIG. 25Ashows animals immunized with VXA-RSV-f before RSV challenge werecompletely protected against RSV replication in lung and nasal samples.Animals immunized with either buffer, adjuvant alone, or FI-RSV were notprotected. Two different doses of vaccine were given for the i.n.treated animals (1×10⁸=low). N=6, except the no infection controlanimals (N=3). In FIG. 25B, inflammation in the lungs was scored byimmunohistology. The vaccine groups (VXA-RSV-f and RSV2) did not causeadaptive immune enhanced inflammation (PB, PA, A, IP) as did the FI-RSVgroup. In FIG. 25C, cytokine abundance levels were measured by qRT-PCR.Only the FI-RSV immunized group had substantial increases in therelative abundance of IL-4 and IL-13, the Th2 cytokines measured.

FIGS. 26A-26C shows oral immunization induces protection against RSVreplication and disease. (Experiment XV-112). In FIG. 26A, animalsimmunized orally show dose dependent immune responses to RSV infection,with the highest dose leading to complete protection in the lungs andalmost complete protection in the nose. In contrast, there was noprotection in the buffer control group. In FIG. 26B, inflammation wascompared between vaccine doses and the buffer control group. In FIG.26C, cytokine abundance levels were measured by qRT-PCR. No group hadsubstantial increases in the relative abundance of either IL-4 or IL-13,the Th2 cytokines measured. (N=8 with the exception of buffer controlN=6).

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that delivery of an immunogenic biologicalagent to a particular part of the small intestine, i.e., the ileum,results in a much greater therapeutic response than when the agent isnot targeted, or is targeted to a different site. This allows for designof more effective vaccines, reduced costs for materials, and reducedside effects for the recipient.

I. DEFINITIONS

The term “immunogenic” refers to the ability of an agent to give rise toan immune response in a host, either humoral or cell-mediated.Immunogenic agents are typically “foreign” to the host, e.g., from adifferent species, or from a bacteria, virus, or fungus. A non-foreignagent can be immunogenic, e.g., in the case of an autoimmune response.Certain cancer cell-specific agents can be exploited as immunogenicagents, allowing the host's immune system to attack the cancer.

The term “biological agent” refers to a nucleic acid, polypeptide,glycoprotein, carbohydrate, lipid, or modified form thereof (e.g.methylated, glycosylated, detectably labeled). Biological agents aredistinguished from small molecule drugs in that they can be created bybiological processes (including recombinant techniques) instead ofchemical synthesis. Biological agents can, however, be chemicallymodified or include non-natural nucleotides or amino acids. Biologicalagents can also be non-naturally occurring, e.g., recombinant orchimeric entities.

As used herein, an “immunogenic biological agent” refers to an agentthat acts directly as an antigen (e.g., is recognized by a T cellreceptor or antibody), or an agent that, once expressed in a cell, actsas an antigen. For example, an immunogenic biological agent can includean expression vector encoding an immunogenic polypeptide.

The term “antigen” refers to a polypeptide, glycoprotein, lipoprotein,lipid, carbohydrate, or other agent that is bound (e.g., recognized as“foreign”) by a T cell receptor and/or antibody. Antigens are commonlyderived from bacterial, viral, or fungal sources. The term “derivedfrom” indicates that the antigen is essentially as it exists in itsnatural antigenic context, or that it has been modified to be expressedunder certain conditions, to include only the most immunogenic portion,or to remove other potentially harmful associated components, etc.

An “immunogenically effective dose or amount” of a composition asdescribed herein is an amount that elicits or modulates an immuneresponse specific for an antigen selected for vaccination. Immuneresponses include humoral immune responses and cell-mediated immuneresponses. An immunogenic composition can be used therapeutically orprophylactically to treat or prevent disease at any stage.

“Humoral immune responses” are mediated by cell free components of theblood, e.g., plasma or serum; transfer of the serum or plasma from oneindividual to another transfers humoral immunity. Humoral immuneresponses are typically B cell-mediated, e.g., antibody production.

“Cell mediated immune responses” are mediated by antigen specificlymphocytes; transfer of the antigen specific lymphocytes from oneindividual to another transfers immunity. Cell-mediated immune responsesare mediated at least in part by T cells, and can be detected, e.g., bydetecting T cell-specific cytokines or increase in T cell proliferation.

The “ileum” is the longest of the three segments that form the smallintestine, along with the duodenum and jejunum. It is makes up theterminal portion, between the jejunum and cecum.

An enteric coating is a barrier applied to oral medications thatprevents the therapeutic agent inside from being digested in the low pHenvironment of the stomach and duodenum (˜pH 3).

A delivery agent, such as an enteric coating, matrix, or capsule, issaid to retain an encompassed or embedded therapeutic agent when atleast 60%, e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% ofthe original administered amount of therapeutic agent remainsencompassed or embedded within the agent. The delivery agent, e.g.,enteric coating or matrix, is typically designed to disintegrate undercertain conditions and release the therapeutic agent. Disintegration canbe gradual, e.g., in the case of a thicker or more chemically complexcoating. The enteric coating is said to “disintegrate” once the coatingthickness is reduced at least 10%, e.g., at least 25%, 50%, or 75%compared to the original administered thickness. Disintegration is notan absolute term, as it can occur over a different time course dependingon conditions. For example, a coating that is designed to disintegratein 5 minutes at pH 6.5 may disintegrate, albeit slowly, at pH 6 (e.g.,in 1 hour). Disintegration does not necessarily indicate that theencompassed or embedded therapeutic agent is released. The therapeuticagent can, however, begin to be released before the enteric coating ormatrix is entirely disintegrated.

The term “chimeric” or “recombinant” as used herein with reference,e.g., to a nucleic acid, protein, or vector, indicates that the nucleicacid, protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein. Thus, for example, chimeric and recombinantvectors include nucleic acid sequences that are not found within thenative (non-chimeric or non-recombinant) form of the vector. A chimericviral expression vector refers to a viral expression vector comprising anucleic acid sequence encoding a heterologous (e.g., immunogenic)polypeptide.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter. Viralexpression vectors are typically rendered replication incompetent orattenuated. A virally-derived vector can include the components of theexpression vector required for expression of a desired sequence, butomit those involved in, e.g., replication or other pathogenic effects.

The terms “promoter” and “expression control sequence” are used hereinto refer to a nucleic acid control sequence that directs transcriptionof a nucleic acid. Promoter sequences are typically near the start siteof transcription, such as a TATA element in the case of a polymerase IItype promoter. A promoter can also include distal enhancer or repressorelements, which can be located as much as several thousand base pairsfrom the start site of transcription. Promoters include constitutive andinducible promoters. A “constitutive” promoter is a promoter that isactive under most environmental and developmental conditions. An“inducible” promoter is a promoter that is active under environmental ordevelopmental regulation. The term “operably linked” refers to afunctional linkage between a nucleic acid expression control sequence(such as a promoter, or array of transcription factor binding sites) anda second nucleic acid sequence, wherein the expression control sequencedirects transcription of the nucleic acid corresponding to the secondsequence.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, heterologous portions ofa protein indicates that the protein comprises two or more subsequencesthat are not found in the same relationship to each other in nature(e.g., a fusion protein). A heterologous nucleic acid or protein is onethat is not found in a particular environment in nature, e.g., aheterologous mouse protein in a human cell.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein to refer to polymers of deoxyribonucleotides or ribonucleotidesin either single- or double-stranded form. The terms encompass genes,cDNA, RNA, and oligonucleotides (short polynucleotides). The termsencompass nucleic acids containing known nucleotide analogs or modifiedbackbone residues or linkages, which are synthetic, naturally occurring,and non-naturally occurring, which have similar binding properties asthe reference nucleic acid, and which are metabolized in a mannersimilar to the reference nucleotides. Examples of such analogs include,without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs). The term “nucleotide” typically refers toa nucleic acid monomer.

Unless otherwise indicated, a particular nucleic acid sequence alsoencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences, as well as thesequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

A “therapeutic dose” or “therapeutically effective amount” or “effectiveamount” of a composition as described herein is an amount that prevents,alleviates, abates, or reduces the severity of symptoms of diseases anddisorders associated with the source of the antigen selected forvaccination (e.g., a virus, bacteria, a parasite, or a cancer).

The term “antibody” refers to a polypeptide encoded by an immunoglobulingene or fragments thereof that specifically bind and recognizes anantigen. Immunoglobulin sequences include the kappa, lambda, alpha,gamma, delta, epsilon, and mu constant region sequences, as well asmyriad immunoglobulin variable region sequences. Light chains areclassified as either kappa or lambda. Heavy chains are classified asgamma, mu, alpha, delta, or epsilon, which in turn define theimmunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

T cells refer to a particular class of lymphocytes that express aspecific receptor (T cell receptor) encoded by a family of genes. Therecognized T cell receptor genes include alpha, beta, delta, and gammaloci, and the T cell receptors typically (but not universally) recognizea combination of MHC plus a short peptide. T cells are typically broadlyclassified as T helper cells (CD4+) and cytotoxic T cells (CD8+).Antibodies are naturally produced by B cells, e.g., Antibody SecretingCells (ASCs). Mature B cells can be naïve, plasma B cells (activated andantibody-producing), memory, B-1, marginal-zone B cells, follicular Bcells, and regulatory B cells.

An adaptive immune response refers to T cell and/or B cell and/orantibody recognition of antigen.

Antigen presenting cells (APCs) are cells that are able to presentimmunogenic peptides or fragments thereof to T cells to activate orenhance an immune response. APCs include dendritic cells, macrophages, Bcells, monocytes and other cells that may be engineered to be efficientAPCs. Such cells may, but need not, be genetically modified to increasethe capacity for presenting the antigen, to improve activation and/ormaintenance of the T cell response, to have anti-tumor effects per seand/or to be immunologically compatible with the receiver (i.e., matchedHLA haplotype). APCs may be isolated from any of a variety of biologicalfluids and organs including bone marrow, peripheral blood, tumor andperitumoral tissues, and may be autologous, allogeneic, syngeneic orxenogeneic cells. APCs typically utilize a receptor from the majorhistocompatability (MHC) locus to present short polypeptides to T cells.

An adjuvant is a non-specific immune response enhancer. Suitableadjuvants include, for example, cholera toxin, monophosphoryl lipid A(MPL), Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, Quil A,and Al(OH). Adjuvants can also be those substances that cause APCactivation and enhanced presentation of T cells through secondarysignaling molecules like Toll-like receptors, e.g., double-stranded RNA(dsRNA), dsRNA mimetics, bacterial flagella, LPS, CpG DNA, and bacteriallipopeptide (Reviewed recently in [Abreu et al., J Immunol, 174(8),4453-4460 (2005)]).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acids. The termsapply to amino acid polymers in which one or more amino acid residue isan artificial chemical mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers andnon-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., a carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine I, Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of complementary (or largelycomplementary) nucleotides in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an individual polypeptide or dsRNA or a portionthereof) or may comprise a variant of such a sequence. Polynucleotidevariants may contain one or more substitutions, additions, deletionsand/or insertions such that at least one biological activity of theencoded polypeptide (e.g., immunogenicity) is not diminished, relativeto a polypeptide comprising native antigens. Polynucleotide variants maycontain one or more substitutions, additions, deletions and/orinsertions such that the adjuvant activity of an encoded dsRNA is notdiminished, relative to a dsRNA that does not contain the substitutions,additions, deletions and/or insertions. Variants preferably exhibit atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a polynucleotidesequence that encodes a native polypeptide or a portion thereof or adsRNA.

The terms “identical” or percent “identity,” in the context of two ormore polynucleotide or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more identity over a specified region), whencompared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. Such sequences are then said to be “substantiallyidentical.” This definition also refers to the compliment of a testpolynucleotide sequence. Optionally, the identity exists over a regionthat is at least about 10 to about 100, about 20 to about 75, about 30to about 50 amino acids or nucleotides in length.

A “control” sample or value refers to a sample that serves as areference, usually a known reference, for comparison to a test sample.For example, a test sample can be taken from a test condition, e.g., inthe presence of a test compound or treatment, and compared to samplesfrom known conditions, e.g., in the absence of the test compound(negative control), or in the presence of a known compound (positivecontrol). In the context of the present disclosure, an example of anegative control would be a biological sample from a known healthy(non-infected) individual, and an example of a positive control would bea biological sample from a known infected patient. A control can alsorepresent an average value or a range gathered from a number of tests orresults. One of skill in the art will recognize that controls can bedesigned for assessment of any number of parameters. For example, acontrol can be devised to compare therapeutic benefit based onpharmacological data (e.g., half-life) or therapeutic measures (e.g.,comparison of benefit and/or side effects). Controls can be designed forin vitro applications. One of skill in the art will understand whichcontrols are valuable in a given situation and be able to analyze databased on comparisons to control values. Controls are also valuable fordetermining the significance of data. For example, if values for a givenparameter are widely variant in controls, variation in test samples willnot be considered as significant.

The term “diagnosis” refers to a relative probability that a subject hasa disorder such as an infection or cancer. Similarly, the term“prognosis” refers to a relative probability that a certain futureoutcome may occur in the subject. The terms are not intended to beabsolute, as will be appreciated by any one of skill in the field ofmedical diagnostics.

The terms “therapy,” “treatment,” and “amelioration” refer to anyreduction in the severity of symptoms. In the context of infection,treatment can refer to a reduction of infectious agent, reducedsymptoms, etc. In the case of treating cancer, treatment can refer to,e.g., reducing tumor size, number of cancer cells, growth rate,metastatic activity, reducing cell death of non-cancer cells, etc. Theterms “treat” and “prevent” are not intended to be absolute terms.Treatment and prevention can refer to any comparative reduction orapparent absence of infectious agent, delay in onset, amelioration ofsymptoms, improvement in patient survival, increase in survival time orrate, etc. Treatment and prevention can be complete (undetectable levelsof infectious agent or neoplastic cells) or partial, such that fewerinfectious agent or neoplastic cells are found in a patient than wouldhave occurred without the presently described immunogenic biologicalagents. The effect of treatment can be compared to an individual or poolof individuals not receiving the treatment, or to the same patient priorto treatment or at a different time during treatment. In some aspects,the severity of infection or disease is reduced by at least 10%, ascompared, e.g., to the individual before administration or to a controlindividual not undergoing treatment. In some aspects the severity ofinfection or disease is reduced by at least 25%, 50%, 75%, 80%, or 90%,or in some cases, no longer detectable using standard diagnostictechniques.

“Subject,” “patient,” “individual” and like terms are usedinterchangeably and refer to, except where indicated, mammals such ashumans and non-human primates, as well as rabbits, rats, mice, goats,pigs, and other mammalian species. The term does not necessarilyindicate that the subject has been diagnosed with a particular disease,but typically refers to an individual under medical supervision. Apatient can be an individual that is seeking treatment, monitoring,adjustment or modification of an existing therapeutic regimen, etc.

II. IMMUNOGENIC BIOLOGICAL AGENTS

An immunogenic biological agent is any biological agent that causes animmune response in the host, e.g., human host. The immunogenicbiological agent can thus be a polypeptide (e.g., glycoprotein,phosphoprotein, or other modified form), carbohydrate, lipid,polynucleotide (e.g., chromatin, methylated polynucleotide, or othermodified form). In some embodiments, the immunogenic biological agentdirectly causes an immune response, e.g., is itself a target immunogen(antigen). In some embodiments, the immunogenic biological agent is apolynucleotide encoding the target immunogen. For example, when apolynucleotide encoding a target antigen is expressed in an antigenpresenting cell (APC), an immune response is mounted against theexpressed antigen. The immunogenic biological agent can be administeredalone, in combination with a second, third, and/or fourth immunogenicbiological agent (e.g., in the case of a multi-target preventativevaccine), and/or in combination with an adjuvant to increase the immuneresponse.

A. Expression Vectors

Expression vectors for use as described herein can includevirally-derived vectors, e.g., recombinant adeno-associated virus (AAV)vectors, retroviral vectors, adenoviral vectors, modified vacciniaAnkara (MVA) vectors, and lentiviral (e.g., HSV-1-derived) vectors (see,e.g., Brouard et al. (2009) British J. Pharm. 157:153). Virally-derivedvectors for therapeutic use are typically rendered replicationincompetent or attenuated. For example, in the case of an adenoviralvector, the adenoviral genome can be modified to remove the E1 and E3genes. For production, the replication deficient vector can beadministered to a cell that expresses the E1 gene such that recombinantadenovirus (rAd) is produced by the cell. This rAd can be harvested andused for a single round of infection to deliver the transgeniccomposition to another cell within a mammal in order to elicit immuneresponses to an encoded polypeptide antigen.

Examples of suitable viral vectors include adenovirus 5, including, forexample, Ad5 with deletions of the E1/E3 regions and Ad5 with a deletionof the E4 region as described in U.S. Pat. No. 8,222,224 and Scallan etal. Clinical and Vaccine Immunology 2013; 20(1): 85-94. An exemplary Ad5viral vector backbone is provided in SEQ ID NO: 7. Other suitableadenoviral vectors include strains 2, orally tested strains 4 and 7,enteric adenoviruses 40 and 41, and other strains (e.g. Ad34, Ad26, orAd35) that are sufficient for delivering an antigen and eliciting anadaptive immune response to the transgene antigen [Lubeck et al., ProcNatl Acad Sci USA, 86(17), 6763-6767 (1989); Shen et al., J Virol,75(9), 4297-4307 (2001); Bailey et al., Virology, 202(2), 695-706(1994)]. The viral vector does not need to have been isolated fromhumans, but can come from a non-human such as chimpanzee adenovirus 3(ChAd3) (see, e.g., Colloca et al. (2012) Sci. Transl. Med. 4:115;Stanley et al. (2014) Nat. Med. doi:10.1038/nm.3702). In someembodiments, the adenoviral vector is a live, replication incompetentadenoviral vector (such as E1 and E3 deleted rAd5), live and attenuatedadenoviral vector (such as the E1B55K deletion viruses), or a liveadenoviral vector with wild-type replication.

Transcriptional and translational control sequences in expressionvectors to be used as described herein can be provided by viral sources.For example, commonly used promoters and enhancers are derived, e.g.,from beta actin, adenovirus, simian virus (SV40), and humancytomegalovirus (CMV). For example, vectors allowing expression ofproteins under the direction of the CMV promoter, SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, transducer promoter, orother promoters shown effective for expression in mammalian cells aresuitable. Additional viral and non-viral promoter, control and/or signalsequences may be used, provided such control sequences are compatiblewith the host cells to be transfected.

B. Immunogens

Immunogens for use as described herein can be derived from antigens,such as, for example, viral antigens, bacterial antigens, cancerantigens, fungal antigens, or parasite antigens (see, e.g., U.S. Pat.No. 8,222,224 for a list of antigens that can be used as describedherein).

Particular examples of antigens that can be used as described herein arethose derived from norovirus (e.g., VP1) and Respiratory syncytial virus(RSV) (e.g.,). Other suitable antigens include those from the influenzavirus (e.g., HA, NA, M1, NP), human immunodeficiency virus (HIV, e.g.,gag, pol, env, etc.), human papilloma virus (HPV, e.g., capsid proteinssuch as L1), Venezuelan Equine Encephalomyelitis (VEE) virus, EpsteinBarr virus, herpes simplex virus (HSV), human herpes virus,rhinoviruses, cocksackieviruses, enteroviruses, hepatitis A, B, C, E,and G (HAV, HBV, HCV, HEV, HGV e.g., surface antigen), mumps virus,rubella virus, measles virus, poliovirus, smallpox virus, rabies virus,and Varicella-zoster virus.

Suitable viral antigens also include viral nonstructural proteins, e.g.,proteins encoded by viral nucleic acid that do not encode for structuralpolypeptides, in contrast to those that make capsid or the proteinsurrounding a virus. Non-structural proteins include those proteins thatpromote viral nucleic acid replication, viral gene expression, orpost-translationsal processing, such as, for example, Nonstructuralproteins 1, 2, 3, and 4 (NS1, NS2, NS3, and NS4, respectively) fromVenezuelan Equine encephalitis (VEE), Eastern Equine Encephalitis (EEE),or Semliki Forest.

Bacterial antigens can be derived from, for example, Staphylococcusaureus, Staphylococcus epidermis, Helicobacter pylori, Streptococcusbovis, Streptococcus pyogenes, Streptococcus pneumoniae, Listeriamonocytogenes, Mycobacterium tuberculosis, Mycobacterium leprae,Corynebacterium diphtheriae, Borrelia burgdorferi, Bacillus anthracis,Bacillus cereus, Clostridium botulinum, Clostridium difficile,Salmonella typhi, Vibrio chloerae, Haemophilus influenzae, Bordetellapertussis, Yersinia pestis, Neisseria gonorrhoeae, Treponema pallidum,Mycoplasm sp., Legionella pneumophila, Rickettsia typhi, Chlamydiatrachomatis, and Shigella dysenteriae, Vibrio cholera (e.g., Choleratoxin subunit B, cholera toxin-coregulated pilus (TCP)); Helicobacterpylorii (e.g., VacA, CagA, NAP, Hsp, catalase, urease); E. coli (e.g.,heat-labile enterotoxin, fimbrial antigens).

Parasite antigens can be derived from, for example, Giardia lamblia,Leishmania sp., Trypanosoma sp., Trichomonas sp., Plasmodium sp. (e.g.,P. falciparum surface protein antigens such as pfs25, pfs28, pfs45,pfs84, pfs 48/45, pfs 230, Pvs25, and Pvs28); Schistosoma sp.;Mycobacterium tuberculosis (e.g., Ag85, MPT64, ESAT-6, CFP10, R8307,MTB-32 MTB-39, CSP, LSA-1, LSA-3, EXP1, SSP-2, SALSA, STARP, GLURP,MSP-1, MSP-2, MSP-3, MSP-4, MSP-5, MSP-8, MSP-9, AMA-1, Type 1 integralmembrane protein, RESA, EBA-175, and DBA).

Fungal antigens can be derived from, for example, Tinea pedis, Tineacorporus, Tinea cruris, Tinea unguium, Cladosporium carionii,Coccidioides immitis, Candida sp., Aspergillus fumigatus, andPneumocystis carinii.

Cancer antigens include, for example, antigens expressed orover-expressed in colon cancer, stomach cancer, pancreatic cancer, lungcancer, ovarian cancer, prostate cancer, breast cancer, skin cancer(e.g., melanoma), leukemia, or lymphoma. Exemplary cancer antigensinclude, for example, HPV L1, HPV L2, HPV E1, HPV E2, placental alkalinephosphatase, AFP, BRCA1, Her2/neu, CA 15-3, CA 19-9, CA-125, CEA, Hcg,urokinase-type plasminogen activator (Upa), plasminogen activatorinhibitor, CD53, CD30, CD25, C5, CD11a, CD33, CD20, ErbB2, CTLA-4. SeeSliwkowski & Mellman (2013) Science 341:6151 for additional cancertargets.

C. Adjuvants

In some embodiments, the compositions further comprise at least oneadjuvant. Suitable adjuvants include, for example, the lipids andnon-lipid compounds, cholera toxin (CT), CT subunit B, CT derivativeCTK63, E. coli heat labile enterotoxin (LT), LT derivative LTK63,Al(OH)₃, and polyionic organic acids as described in e.g.,WO2004/020592, Anderson and Crowle, Infect. Immun. 31(1):413-418 (1981),Roterman et al., J. Physiol. Pharmacol., 44(3):213-32 (1993), Arora andCrowle, J. Reticuloendothel. 24(3):271-86 (1978), and Crowle and May,Infect. Immun. 38(3):932-7 (1982)). Suitable polyionic organic acidsinclude for example,6,6′-[3,3′-demithyl[1,1′-biphenyl]-4,4′-diyl]bis(azo)bis[4-amino-5-hydrox-y-1,3-naphthalene-disulfonicacid] (Evans Blue) and 3,3′-[1,1′biphenyl]-4,4′-diylbis(azo)bis[4-amino-1-naphthalenesulfonic acid](Congo Red). It will be appreciated by those of skill in the art thatthe polyionic organic acids may be used for any nucleic acid-basedvaccination method in conjunction with any type of administration.

TLR-3 agonists (e.g., dsRNA, and mimetics thereof such as polyI:C, polyA:U, and polyI:polyC) can also be used. TLR-3 agonists include, forexample, short hairpin RNA, virally derived RNA, short segments of RNAthat can form double-strands or short hairpin RNA, and short interferingRNA (siRNA). In some embodiments, the TLR-3 agonist is virally deriveddsRNA, e.g., dsRNA derived from a Sindbis virus or dsRNA viralintermediates (Alexopoulou et al. (2001) Nature 413:732). In someembodiments, the TLR-3 agonist is a short hairpin RNA. Short hairpin RNAsequences typically comprise two complementary sequences joined by alinker sequence. The particular linker sequence is not a critical aspectof the invention. Any appropriate linker sequence can be used so long asit does not interfere with the binding of the two complementarysequences to form a dsRNA. TLR-3 agonists can result in pro-inflammatorycytokine release (e.g. IL-6, IL-8, TNF-alpha, IFN-alpha, IFN-beta) whencontacted with a responder cell (e.g., a dendritic cell, a peripheralblood mononuclear cell, or a macrophage) in vitro or in-vivo.

Other suitable adjuvants include topical immunomodulators such as,members of the imidazoquinoline family such as, for example, imiquimodand resiquimod (see, e.g., Hengge et al., Lancet Infect. Dis.1(3):189-98 (2001).

Additional suitable adjuvants are commercially available as, forexample, additional alum-based adjuvants (e.g., Alhydrogel, Rehydragel,aluminum phosphate, Algammulin); oil based adjuvants (Freund'sIncomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,Mich.), Specol, RIBI, TiterMax, Montanide ISA50 or Seppic MONTANIDE ISA720); nonionic block copolymer-based adjuvants, cytokines (e.g., GM-CSFor Flat3-ligand); Merck Adjuvant 65 (Merck and Company, Inc., Rahway,N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); salts of calcium,iron or zinc; an insoluble suspension of acylated tyrosine; acylatedsugars; cationically or anionically derivatized polysaccharides;polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A andQuil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, are alsosuitable adjuvants. Hemocyanins (e.g., keyhole limpet hemocyanin) andhemoerythrins can also be used as adjuvants. Polysaccharide adjuvantssuch as, for example, chitin, chitosan, and deacetylated chitin are alsosuitable as adjuvants. Other suitable adjuvants include muramyldipeptide (MDP, N acetylmuramyl L alanyl D isoglutamine) bacterialpeptidoglycans and their derivatives (e.g., threonyl-MDP, and MTPPE).BCG and BCG cell wall skeleton (CWS) can be used as adjuvants, with orwithout trehalose dimycolate. Trehalose dimycolate can be used itself(see, e.g., U.S. Pat. No. 4,579,945). Detoxified endotoxins are alsouseful as adjuvants alone or in combination with other adjuvants (see,e.g., U.S. Pat. Nos. 4,866,034; 4,435,386; 4,505,899; 4,436,727;4,436,728; 4,505,900; and 4,520,019). The saponins QS21, QS17, QS7 arealso useful as adjuvants (see, e.g., U.S. Pat. No. 5,057,540; EP 0362279; WO 96/33739; and WO 96/11711). Other suitable adjuvants includeMontanide ISA 720 (Seppic, France), SAF (Chiron, Calif, United States),ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g.,SBAS-2, SBAS-4 or SBAS-6 or variants thereof, available from SmithKlineBeecham, Rixensart, Belgium), Detox (Corixa, Hamilton, Mont.), andRC-529 (Corixa, Hamilton, Mont.).

Superantigens are also contemplated for use as adjuvants in the presentinvention. Superantigens include Staphylococcus exoproteins, such as thealpha, beta, gamma, and delta enterotoxins from S. aureus and S.epidermidis, and the alpha, beta, gamma, and delta E. coli exotoxins.Common Staphylococcus enterotoxins are known as staphylococcalenterotoxin A (SEA) and staphylococcal enterotoxin B (SEB), withenterotoxins through E (SEE) being described (Rott et al., 1992).Streptococcus pyogenes B (SEB), Clostridium perfringens enterotoxin(Bowness et al., 1992), cytoplasmic membrane-associated protein (CAP)from S. pyogenes (Sato et al., 1994) and toxic shock syndrome toxin 1(TSST 1) from S. aureus (Schwab et al., 1993) can also be used.

For the pharmaceutical compositions provided herein, the adjuvant(s) canbe designed to induce, e.g., an immune response predominantly of the Th1or Th2 type. High levels of Th1-type cytokines (e.g., IFN-gamma,TNF-alpha, IL-2 and IL-12) tend to favor the induction of cell mediatedimmune responses to an administered antigen. In contrast, high levels ofTh2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor theinduction of humoral immune responses. Following oral delivery of acomposition comprising an immunogenic polypeptide as provided herein, animmune response that includes Th1- and Th2-type responses will typicallybe elicited.

III. TARGETED DELIVERY SYSTEMS

The presently described compositions and methods for ileal delivery canrely on appropriate coatings, matrices, and devices such as thosedescribed below.

A. Enteric Coatings, Matrices, and Devices

Enteric coatings are used to shield substances from the low pHenvironment of the stomach and delay release of the enclosed substanceuntil it reaches a desired target later in the digestive tract. Entericcoatings are known, and commercially available. Examples includepH-sensitive polymers, bio-degradable polymers, hydrogels, time-releasesystems, and osmotic delivery systems (see, e.g., Chourasia & Jain(2003) J. Pharm. Pharmaceutical Sci. 6:33).

The pH of the gastrointestinal tract (GIT) progresses from very acidicin the stomach (pH ˜2), to more neutral in the ileum (pH ˜5.8-7.0). pHsensitive coatings can be used that dissolve in the ileum or just beforethe ileum. Examples include Eudragit® L and S polymers (threshold pH'sranging from 5.5-7.0); polyvinyl acetate phthalate (pH 5.0),hydroxypropyl methylcellulose phthalate 50 and 55 (pH 5.2 and 5.4,respectively), and cellulose acetate phthalate (pH 5.0). Thakral et al.(2013) Expert Opin. Drug Deliv. 10:131 review Euragit® formulations forileal delivery, in particular, combinations of L and S that ensuredelivery at pH≤7.0. Crotts et al. (2001) Eur. J Pharm. Biol. 51:71describe Eudragit® formulations with appropriate disintegrationproperties. Vijay et al. (2010) J. Mater. Sci. Mater. Med. 21:2583review acrylic acid (AA)-methyl methacrylate (MMA) based copolymers forileal delivery at pH 6.8.

For ileal delivery, the polymer coating typically dissolves at about pH6.8 and allows complete release within about 40 min (see, e.g.,Huyghebaert et al. (2005) Int. J. Pharm. 298:26). To accomplish this, atherapeutic substance can be covered in layers of different coatings,e.g., so that the outermost layer protects the substance through low pHconditions and is dissolved when the tablet leaves the stomach, and atleast one inner layer that dissolves as the tablet passes intoincreasing pH. Examples of layered coatings for delivery to the distalileum are described, e.g., in WO2013148258.

Biodegradable polymers (e.g., pectin, azo polymers) typically rely onthe enzymatic activity of microflora living in the GIT. The ileumharbors larger numbers of bacteria than earlier stages, includinglactobacilli and enterobacteria.

Osmotic-controlled Release Oral delivery Systems (OROS®; Alza) is anexample of an osmotic system that degrades over time in aqueousconditions. Such materials can be manipulated with other coatings, or invarying thicknesses, to deliver specifically to the ileum (see, e.g.,Conley et al. (2006) Curr. Med. Res. Opin. 22:1879).

Combination polymers for delivery to the ileum are reported inWO2000062820. Examples include Eudragit® L100-55 (25 mg/capsule) withtriethyl citrate (2.4 mg/capsule), and Povidone K-25 (20 mg/tablet)followed by Eudragit® FS30D (30 mg/tablet). pH sensitive polymers can beapplied to effect delivery to the ileum, as described above and, e.g.,methacrylic acid copolymers (e.g., poly(methacrylic acid-co-methylmethacrylate) 1:1), cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropyl methylcellulose acetatesuccinate, polyvinyl acetate phthalate, cellulose acetate trimellitate,carboxymethyl ethyl-cellulose, shellac or other suitable polymer(s). Thecoating layer can also be composed of film-forming polymers beingsensitive to other luminal components than pH, such as bacterialdegradation or a component that has such a sensitivity when it is mixedwith another film-forming polymer. Examples of such components providingdelayed release to the ileum are polymers comprising azo bond(s),polysaccharides such as pectin and its salts, galactomannans, amyloseand chondroitin, disulphide polymers and glycosides.

Components with varying pH, water, and enzymatic sensitivities can beused in combination to target a therapeutic composition to the ileum.The thickness of the coating can also be used to control release. Thecomponents can also be used to form a matrix, in which the therapeuticcomposition is embedded. See generally, Frontiers in Drug Design &Discovery (Bentham Science Pub. 2009) vol. 4.

B. Frequency or Radio-Controlled Capsules

As an alternative to dissolving coatings and matrices, site-specificdelivery can be via capsules that release upon an externally generatedsignal. Early models released for a high-frequency (HF) signal, asdisclosed in Digenis et al. (1998) Pharm. Sci. Tech. Today 1:160. Theoriginal HF capsule concept has since been updated and the resultmarketed as InteliSite®. The updated capsule is a radio-frequencyactivated, non-disintegrating delivery system. Radiolabeling of thecapsule permits the determination of the capsule location within aspecific region of the GI tract via gamma scintigraphy. When the capsulereaches the desired location in the GI tract, external activation opensa series of windows to the capsule drug reservoir.

In some embodiments, the immunogenic biological agent can be enclosed ina radio-controlled capsule, so that the capsule is tracked and signaledonce it reaches the ileum. In some embodiments, the capsule is signaledat a given time after administration that corresponds to when thecapsule is expected to arrive in the ileum, with or without detecting.

C. Formulations

Pharmaceutical compositions can be used for prophylactic and therapeuticpurposes as described herein. As explained above, pharmaceuticalcompositions can be prepared to protect against stomach degradation suchthat the administered immunogenic biological agent reach the desiredlocation. Methods for microencapsulation of DNA and drugs for oraldelivery are described, e.g., in US2004043952.

An immunogenic pharmaceutical composition can contain pharmaceuticallyacceptable salts of the immunogenic biological agent (e.g., immunogenicpolypeptide, or polynucleotide encoding an immunogenic polypeptide).Such salts may be prepared from pharmaceutically acceptable non-toxicbases, including organic bases (e.g., salts of primary, secondary andtertiary amines and basic amino acids) and inorganic bases (e.g.,sodium, potassium, lithium, ammonium, calcium and magnesium salts). Someparticular examples of salts include phosphate buffered saline andsaline (e.g., for ingestion, nasal delivery, or injection).

A delayed release coating or an additional coating of the formulationcan contain other film-forming polymers being non-sensitive to luminalconditions for technical reasons or chronographic control of the drugrelease. Materials to be used for such purpose includes, but are notlimited to; sugar, polyethylene glycol, polyvinylpyrrolidone, polyvinylalcohol, polyvinyl acetate, hydroxypropyl cellulose, methylcellulose,ethylcellulose, hydroxypropyl methylcellulose, carboxymethylcellulosesodium and others, used alone or in mixtures.

Additives such as dispersants, colorants, pigments, additional polymers,e.g., poly(ethylacrylat, methylmethacrylat), anti-tacking andanti-foaming agents can be included into a coating layer. Othercompounds may be added to increase film thickness and to decreasediffusion of acidic gastric juices into the core material. The coatinglayers can also contain pharmaceutically acceptable plasticizers toobtain desired mechanical properties. Such plasticizers are forinstance, but not restricted to, triacetin, citric acid esters, phthalicacid esters, dibutyl sebacate, cetyl alcohol, polyethylene glycols,glycerol monoesters, polysorbates or other plasticizers and mixturesthereof. The amount of plasticizer can be optimised for each formula,and in relation to the selected polymer(s), selected plasticizer(s) andthe applied amount of said polymer(s).

Other suitable pharmaceutical ingredients known in the art can beemployed in the pharmaceutical compositions of this invention. Suitablecarriers include, for example, water, saline, alcohol, a fat, a wax, abuffer, a solid carrier, such as mannitol, lactose, starch, magnesiumstearate, sodium saccharine, talcum, cellulose, glucose, sucrose, andmagnesium carbonate, or biodegradable microspheres (e.g., polylactatepolyglycolate). Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128;5,820,883. The immunogenic polypeptide and/or carrier expression vectorcan be encapsulated within the biodegradable microsphere or associatedwith the surface of the microsphere.

Such compositions may also comprise non-immunogenic buffers (e.g.,neutral buffered saline or phosphate buffered saline), carbohydrates(e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins,polypeptides or amino acids such as glycine, antioxidants,bacteriostats, chelating agents such as EDTA or glutathione, adjuvants(e.g., aluminum hydroxide), suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilate. Compounds may also be encapsulated withinliposomes using well known technology.

IV. IMMUNE RESPONSES AND VACCINES

The pharmaceutical compositions for ileum delivery as described hereinare designed to elicit an immune response from an individual that isspecific for an immunogenic biological agent included in thepharmaceutical composition. The pharmaceutical composition can be usedprophylactically or therapeutically as a vaccine to avoid or reduce aviral infection, bacterial infection, parasitic infection, fungalinfection, or cancer. The pharmaceutical compositions can be used totreat at any stage, e.g., at the pre-cancer, cancer, or metastaticstages, or to prevent disease or infection.

For example, the compositions described herein may be used to prevent ortreat infection, such as influenza, hepatitis, or HIV, or for preventionor treatment of cancer. Within such methods, pharmaceutical compositionsare typically administered to an individual that may or may not beafflicted with the disease, disorder, or infection. In some embodiments,a disease, disorder, or infection is diagnosed prior to administration,e.g., using criteria generally accepted in the art. For example, viralinfection may be diagnosed by the measurement of viral titer in a samplefrom the patient, bacterial infection may be diagnosed by detecting thebacteria in a sample from the patient, and cancer may be diagnosed bydetecting the presence of a malignant tumor. Pharmaceutical compositionscan be administered either prior to or following surgical removal ofprimary tumors and/or treatment such as administration of radiotherapyor conventional chemotherapeutic drugs.

Immunotherapy is typically active immunotherapy, in which treatmentrelies on the in vivo stimulation of the endogenous host immune systemto react against, e.g., tumors or bacterially or virally infected cells,with the administration of immune response-modifying agents (e.g.,immunogenic biological agents).

Frequency of administration of the prophylactic or therapeuticcompositions described herein, as well as dosage, will vary fromindividual to individual, and can be readily established using standardtechniques. Typically, between 1 and 52 doses can be administered over a52 week period. In some embodiments, 3 doses are administered, atintervals of 1 month, or 2-3 doses are administered every 2-3 months. Insome embodiments, a combination of more than one antigen can beadministered simultaneously or sequentially, e.g., an annual influenzavaccine that contains individual components directed at each subtype ofinfluenza or multiple clades within a subtype. In some embodiments, theintervals are more like once a year, e.g., an annual flu vaccine basedon the particular current strain. Booster vaccinations can be givenperiodically thereafter. Alternate protocols may be appropriate forindividual patients and particular diseases and disorders.

A suitable dose is an amount of an immunogenic biological agent that,when administered as described above, is capable of promoting, e.g., ananti-tumor, an anti-viral, or an antibacterial, immune response, and isat least 15-50% above the basal (untreated) level, or at least 5-50%(e.g., 5%, 10%, 20%, 30%, 50%, 1.5-fold, 2-fold, or higher) above thelevel from non-ileum targeted treatment. Such response can be monitoredby measuring the anti-tumor antibodies in a patient or byvaccine-dependent generation of cytolytic T cells capable of killing,e.g., the patient's tumor cells, the patient's virally infected cells,or the patient's bacterially infected cells in vitro. Such vaccines canalso generate an immune response that leads to an improved clinicaloutcome (e.g., complete or partial or longer disease-free survival,reduced viral titers) in vaccinated patients as compared tonon-vaccinated patients, or patients receiving non-ileum targetedtreatment.

In general, an appropriate dosage and treatment regimen provides theactive compound(s) in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome (e.g., reduced or negative viral titer,more frequent remissions, complete or partial, or longer disease-freesurvival) in treated patients as compared to patients treated withnon-ileum targeted treatment, or non-treated patients. Such immuneresponses can generally be evaluated using standard proliferation,cytotoxicity or cytokine assays described above, which can be performedusing samples obtained from a patient before and after treatment.

For example, detection of immunocomplexes formed between an immunogenicpolypeptide and antibodies in body fluid that are specific for theimmunogenic polypeptide can be used to monitor the effectiveness oftherapy, e.g., for a disease or disorder in which the immunogenicpolypeptide is associated. Samples of body fluid taken from anindividual prior to and subsequent to initiation of therapy (e.g.,ileum-targeted therapy) may be analyzed for the immunocomplexes usingknown methods. Briefly, the number of immunocomplexes detected in bothsamples is compared. A significant change in the number ofimmunocomplexes in the second sample (post-targeted therapy) relative tothe first sample (pre-targeted therapy) reflects successful therapy.

V. EXAMPLES

Pharmaceutical methods for delivering small molecules to the intestineare known, but the ability to deliver a large biological to theintestine for proper immune recognition is poorly understood. Mice arenot able to swallow pills, so it is difficult to perform studies withtablets in animal models. Further, the location of the best place todeliver the vaccine vector in order to elicit a response to transgeneantigen has not been characterized in humans. In sheep, the jejunum wasshown to be the most effective target for eliciting an immune responseto an adenovirally-encoded transgene antigen (Mutwari et al. (1999)Immunology 97:455). Here we show the result of several human ornon-human primate studies with improved human oral dosage forms fordelivery of biological agents.

Example 1

In order to determine which region of the small intestine is most activefor inducing an immune response to antigen, tests were performed inhumans. Radio-controlled capsules were given to healthy normalvolunteers, with the vaccine either released early in the smallintestine (jejunum) or later in the small intestine (ileum). The use ofthe radio-controlled capsules for delivery of small molecule drugs hasbeen described, but not for vaccine delivery (Digenis et al. (1991)Crit. Rev. Ther. Drug Carrier Syst. 7:309).

The vaccine was composed of recombinant adenovirus expressing theinfluenza antigen HA from A/CA/04/2009 (rAd-HA-dsRNA) (see, e.g.,US2012/0244185). A total of 10¹¹ infectious units (IU) were given toeach subject on day 0. The numbers of circulating pre-plasma B cells inperipheral blood were measured by Antibody Secreting Cell (ASC) assay ondays 0 and 7 after the administration of the vaccine. Results onlymeasure the numbers of ASCs that recognize the antigen HA.

Results show that ASCs could be measured 7 days after immunization ineach of the treated groups (FIG. 1 ). Average responses were higher inthe ileum dosed group than the jejunum dosed group. Background ASCs onday 0 were negligible. For the ileum, an average of 340+/−111 (standarderror) IgG and 74+/−18 IgA ASCs were observed on day 7. For the jejunum,the average and standard error responses were 118+/−30 IgG and 28+/−8IgA ASCs. The ileum group was significantly different than placebo(P=0.03 on day 7 for IgA ASC, and trended higher for IgG ASC p=0.07).Contrary to the results in sheep, the results in humans indicate thatileum delivery is more potent at eliciting an IgG or an IgA antibodyresponse than jejunum delivery.

T cell responses were also determined by detecting interferon-y release(IFN-y) using the ELISPOT® assay. FIG. 2 shows that 12/12 of theileum-dosed group had increased levels of IFN-γ, compared to 8/12 of thejejunum-dosed group 7 days post-administration. In addition, IFN-γlevels were significantly higher in the ileum-dosed group than in thejejunum-dosed group.

Microneutralizing (MN) antibody titers to influenza A/CA/07/2009 weremeasured. Increased MN antibody levels are indicative of a neutralizingantibody response. After excluding subjects that had an initialneutralizing antibody response greater than 40 (Faix et al. (2012) PloSOne 7:e34581), the fold increases in MN titers were plotted forindividual subjects. The number of subjects with a positive increase was9 out of 10 for the ileum delivered vaccine versus 6 out of 10 forjejunum delivered vaccine (FIG. 3 ). The geometric mean titers (GMT)were similar between the two groups, with ileum GMT rising from 22 to 92versus the jejunum GMT rising from 18 to 90. The results indicate thatileum release is more reliable at inducing neutralizing antibodyresponses to influenza, possibly leading to a greater percentage ofsubjects protected against influenza.

Example 2

Tablets were hand made using microcrystalline cellulose (PH-101, FMC)and starch (Starch 1500, Colorcon) incorporating 10% barium sulfate as aradiopaque material containing fumed silica as a flow aid and magnesiumstearate as a tablet lubricant. The tablets of 7.14 mm diameter and 150mg weight were coated with Eudragit® L-100 in a pan coater using 10%coating solids weight gain as a guide to whether the enteric coating wasadded; coating solids contained 4 parts Eudragit® polymer to one parttriethyl citrate and 1 part talc. As an initial test of enteric coatingperformance, four cynomolgus macaques were given tablets using an oralgastric tube. The oral gastric tube is solid and rigid, but hollow downthe middle for instilling liquids. It has a flexible silicone tube onthe leading end of the rigid tube that can hold a small tablet in place.The tube and pill apparatus were threaded down the esophagus ofrestrained monkeys until the leading end passed through the cardiacsphincter and into the stomach. A flush of orange juice was used todislodge the pill into the stomach. X-rays were taken at set timepoints, and examined for location and dissolution of the tablet. Table 1summarizes the results.

TABLE 1 L-100 coating performance Time and Pill Location Animal l hr 2hr 3 hr 4 hr 1 stomach stomach stomach intestine 2 stomach intestineintestine dissolved 3 intestine intestine dissolved dissolved 4 stomachstomach intestine dissolved

FIG. 4 shows that the tablets were completely intact in the low pHenvironment of the stomach; there was no evidence of prematuredissolution of the tablets. While large for a monkey, the tablets wereable to pass through the stomach intact into the intestine. In theintestine, they dissolved at a reasonable rate and were completelydissolved in 3 out of 4 monkeys. In the 4^(th) monkey, the pill left thestomach sometime after 3 hours and the pill had not dissolved at thetime of the last x-ray. Overall, the tablets performed in an acceptablemanner and the Eudragit® L-100 coating was selected for future humanstudies.

Example 3

A phase 1, sequentially enrolled clinical study, with a randomized andplacebo-controlled cohort to evaluate safety, and immunogenicity of arecombinant Ad serotype 5 (rAd5) based oral vaccine against H1 seasonalinfluenza was completed. The rAd5 vector (rAd-HA-dsRNA with HA fromA/CA/04/2009) was described in Example 1. The study had an active phaseof approximately 3 months, and was conducted in accordance withapplicable Good Clinical Practice guidelines, the United States Code ofFederal Regulations, and the International Conference on Harmonizationguidelines. Informed consent was obtained from all subjects afterdiscussion of the risks. IRB approval was given before dosing ofsubjects.

Good manufacturing practice (GMP)-grade rAd-HA-dsRNA was produced inWave® bags (GE Healthcare, Waukesha, Wis.) at Lonza Biologicals(Houston, Tex.). Purification was performed by ion exchangechromatography, followed by buffer exchange. Purified vector was mixedwith excipients, lyophilized, and then tableted at Lonza usingmicrocrystalline cellulose and starch as tableting bulk. Tablets wereenteric coated with Eudragit® L 100 (Evonik Industries, Darmstadt,Germany) using a Vector HiCoater® LDCS-5 coater (Vector Freund, CedarRapids, Iowa). The final product was released in one lot, and titered bystandard IU assay. Placebo was prepared as similarly sized and shapedtablets containing 150 mg of microcrystalline cellulose, without entericcoating. The study compared 10⁹ IU, 10¹⁰ IU, and placebo treatedsubjects for the ability to elicit an immune response to transgene.Subjects were given tablets on days 0 and 28.

The numbers of circulating pre-plasma B cells in peripheral blood weremeasured by ASC assays on days 0 and 7 after the initial dose, and atdays 28 and 35 after the second dose (the second dose was delivered atday 28). Results show that ASC counts could be measured 7 days aftereach immunization in the treated groups, but not the placebo group (FIG.5 ). Average responses were higher on day 7, and higher in the high dosegroup than the low dose group. Background ASCs on days 0 and 28 werenegligible, and negligible for the placebo group at all time points. Forthe high dose group, an average of 105+/−33 and 27+/−12 ASCs were foundfor days 7 and 35 respectively. For the low dose group, average ASCswere 41+/−32 and 14+/−8 for days 7 and 35 respectively. The placebogroup had an average of 0.3+/−0.3 and 0, for days 7 and 35 respectively.The high dose group was significantly higher than placebo (P=0.01 and0.05 for days 7 and 35 respectively.)

Neutralizing antibody responses to influenza were measured by MN assay.Results show a dose dependent increase in the MN titers in the treatedgroups versus the placebo control (FIG. 6 ). The frequency of MNresponders with at least a 2-fold increase in the high dose group wassignificantly different than the placebo group (P=0.003 by Fisher'sexact test), whereas the low dose trended higher, but was notsignificantly higher than placebo (P=0.2). After removing subjects thathad MN titers greater than 40, the geometric mean titers (GMT) werecalculated in the remaining subjects (Table 2). Day 56 Geometric FoldTiter Response (GMFR) was also calculated (Table 2). These results showthat neutralizing antibody titers to influenza are being generated byoral immunization, with a greater than 3 fold increase in the GMT afterimmunization in the high dose group. These results show that L 100coated tablets can be used for vaccine delivery to the intestine.

TABLE 2 GMT changes in MN titers for subjects with MN ≤ 40 Group N GMTDO GMT D28 GMT D56 GMFR Placebo 8 14.1 14.1 14.1 1 Low Dose 10 12.3 14.116.2 1.3 High Dose 7 15.6 36.2 53.8 3.4

Example 4

We tested parameters for enteric coatings in vitro to determinedissolution times with varying pH and coating percentage. The dataprovide guidelines for ileal delivery following gastric exposure at lowpH (as in the stomach) and subsequent transit through an increasing pHgradient (as is found in the duodenum and jejunum) prior to reaching theileum.

Tablet disintegration was tested with 150 mg tablets prepared asdescribed above, and coated with 8, 10, or 12% total solids weight gain,utilizing Eudragit® L100, Eudragit® L100-55, or 1:1 (w/w) mixture ofL100 and L100-55 polymers, applied as an organic solvent suspension. Induplicate, tablets prepared with each coating polymer, and at each levelof coating application, were pre-exposed to USP simulated gastric fluid(SGF, pH 1.6, no pepsin) for 120 minutes in a VanKel Bio-Dis IIIreciprocating cylinder dissolution test apparatus at 37° C. at areciprocation rate of 10 dips per minute (DPM). The tablets were thentransferred to USP simulated intestinal fluid (SIF, pH 6.8, nopancreatin). Tablets were observed for disintegration and the time tocomplete disintegration of both tablets was recorded to the nearest 5minutes. The data indicate that disintegration time is influenced byboth polymer composition and thickness and provide guidance with regardto proper selection of coating composition to influence the behavior ofthe coatings after tablets exit the stomach.

Time to disintegrate at indicated coating level (minutes) CoatingPolymer 8% 10% 12% L100 20 30 45 L100/L100-55 15 20 30 L100-55 10 20 25

The effect of pH on disintegration time was tested with 150 mg tablets,coated to 10% total solids weight gain with either Eudragit® L100 orEudragit® L100-55. A series of buffers were prepared by adjusting the pHof USP SIF (no pancreatin) to values encompassing the USP specificationof 6.8. Tablets were pre-exposed to USP SGF (no pepsin) for 120 minutes37° C. and 10 DPM, then transferred to the pH-modified USP SIFsolutions. The tablets were observed for disintegration and the time tocomplete disintegration was recorded to the nearest 5 minutes. The dataindicate that the rate of disintegration is influenced by theenvironmental pH and differs between the two polymers. Again, theresults can be used for proper selection of a coating composition toaccomplish drug retention through the stomach and upper small intestine.

Time to disintegrate for polymer at pH (minutes) pH of SIF Eudragit L100Eudragit L100-55 5.4 250 145 6 110 60 6.4 55 45 7 30 20

Example 5

We carried out a Phase 1, sequentially enrolled study, with a randomizedand placebo-controlled cohort to evaluate safety, and immunogenicity ofa recombinant Ad serotype 5 (rAd5) based oral vaccine against H1seasonal influenza. Tablets containing the vaccine were coated asdescribed herein to dissolve in the ileum. The data show that an oraltablet vaccine would be competitive with existing vaccines in terms ofeliciting neutralizing antibody responses to influenza.

Hemagglutination Inhibition (HAI) responses were measured on days 0 and28 (FIG. 7A). No placebo treated subject seroconverted, but one placebosubject slipped through screening and had a high day 0 value. None ofthe vaccine subjects had a starting HAI titer >20. After immunization,nine subjects in the vaccine group reached seroprotective levels (HAI≥40) (FIG. 7A). The Geometric Mean Titer (GMT) for the group was 61·1(95% CI: 30-124), a 7.7-fold geometric mean fold rise (GMFR) over theinitial GMT of 7·9 (95% CI: 6-11). Of the eleven 4-fold risers (92%),nine seroconverted (SC) with the other 2 subjects showing a 4-foldincrease in HAI titer from 5 to 20. The vaccine group had astatistically significant increase in the number of 4-fold respondersversus placebo (11 versus 0, with P<0·0000 by Fisher's Exact Test). Theplacebo subjects had a GMT of 11.9 (95% CI: 6-25) on day 28 versus a GMTof 11.0 on day 0 (95% CI: 5-23).

Durability of the antibody response was measured by examining HAIresponses 180 days after immunization. In the vaccine-immunized group,75% (9 of 12) of the subjects were seroprotected on day 28 and 75% (9 of12) were still seroprotected on day 180. The HAI GMT were plotted (FIG.7B), and the decrease in the GMT was found to be 28% between 28 and 180days post immunization.

Neutralizing antibody responses to influenza were measured by MN assay.Significant increases in the MN titers in the treated group versus theplacebo control were observed (FIG. 7C). The frequency of 4-fold MNresponders in the vaccine treated group was significantly different thanthe placebo group, with 11 subjects responding in the vaccine treatedgroup versus 0 in the placebo group (P<0·0000 by Fisher's exact test).

After removing subjects that had baseline MN titers (and HAI titers)greater than 40, the geometric mean titers (GMT) were calculated in theremaining subjects on days 0 and 28 as shown in the following table. TheGMT for the vaccine group rose to 247 (95 CI: 89-685) versus no rise inthe placebo for a day 28 GMT of 9.6 (95 CI: 5-18). These calculationshad no impact on the vaccine group, as none of the subjects had highinitial MN or HAI titers. These results show that neutralizing antibodytiters to influenza are generated by oral immunization, with a greaterthan 20-fold increase in the GMT after immunization in thevaccine-treated group.

TABLE 3 GMT changes in HAI and MN titers for subjects with MN ≤ 40.ASSAY GROUP N GMT D0 GMT D28 GMFR SC HAI Placebo 11 8.3 8.8 1.1  0%Vaccine 12 7.9 61.1 7.7 75% MN Placebo 9 9.3 9.6 1.0 N/A Vaccine 12 8.6247 29 N/A

In order to measure total antibody responses to HA, the numbers ofcirculating pre-plasma B cells in peripheral blood were measured by ASCassay on days 0 and 7 after immunization. Results show that ASCs couldbe reliably measured on day 7 in the vaccine-treated group (FIG. 7D).Background ASCs were generally negligible on day 0. For the vaccinetreated group, an average of 992 (+/−std err 209, 95% CI: 532-1452) IgGASCs and 337 IgA ASCs (+/−std err 104, 95% CI: 117-580) each per 1×10⁶PBMC were found for day 7, with only one subject out of 12 having nodetectable ASC response. The placebo group had no IgA spots on day 7,but one subject had a high background smear and a measurable IgG ASCresponse with smaller spots than normally observed. The treated groupwas significantly different than placebo in terms of the ability toelicit an IgG or an IgA ASC response at day 7 (P=0 0007 and P=0.008respectively by T Test).

Subjects were retrospectively measured for their anti-vector titers pre-and post-immunization. Following oral immunization, a fewvaccine-treated subjects had an increase in neutralizing antibodyresponses to Ad5, which led to a 2·6-fold increase in the GMneutralizing antibody titers, compared to 1·0-fold GM fold rise in theplacebo treated subjects. In the vaccine group, HAI and MN responsestrended similarly for individual subjects. Eight subjects were Ad5negative before immunization, and four were Ad5 positive beforeimmunization. One subject that was Ad5 positive did not HAI seroconvert,however, one subject that was Ad5 positive had the highest increase inHAI titers (64 fold) of any of the subjects in the study. This samesubject had a gain in MN titers of 362 fold without any increase in theAd5 neutralizing antibody titers pre and post immunization. There was noobserved correlation between starting Ad5 titers versus fold MN response(or HAI response) for the subjects immunized with the tablet vaccine.

Moreover, the presently disclosed tablet vaccine is stable at roomtemperature for greater than 270 days and can tolerate short-termexcursions at higher temperatures, which makes this approach technicallyfeasible.

Example 5 Discussion

The US military conducted an independent study to measure the effects oftheir seasonal vaccine campaigns on neutralizing antibody responses inmilitary personnel, and reported a MN Titer GMFR of 5.6 after trivalentinactivated vaccine (TIV) injection and a GMFR of 2.2 followinglive-attenuated influenza vaccine (LAIV) intranasal administration,after accounting for subjects that had MN titers above 40 to start (Faixet al. (2012) PloS one 7:e34581). In another study, the SC rate to H1N1was found to be 45% for one injection of 45 ug of HA protein (withoutadjuvant) (Gordon et al. (2012) Vaccine 30:5407), while in another, theH1N1 vaccine was highly immunogenic with a 78% SC rate observed after 1dose of a split vaccine (Greenberg et al. (2009) 361:2405).

In contrast to the variable results observed with injected vaccines, inthe present study, MN GMFR was calculated at 29 for the 12 vaccinetreated subjects with 92% of subjects showing a greater than 4-fold risein MN titers. In the present tablet study, the HAI SC rate among vaccinetreated subjects was 75% with over 92% of subjects having a 4-fold risein HAI titers (FIG. 7A). MN titers were higher than the HAI titers. Itis possible that the MN assay is more sensitive or that the oral rAdbased vaccine elicits stronger neutralizing responses outside the headregion than protein injected vaccines.

HAI responses are elicited with injected commercial vaccines, but HAItiters are known to wane. For example, non-HIV infected volunteers had a67% drop in GMT HAI titers between 1 and 6 months post immunization(Crum-Cianflone et al. (2011) Vaccine 29:3183). Similarly, thepercentage of seroprotected subjects dropped from 75% to 56% for HIVnegative subjects that enrolled with seronegative HAI titers (≤1:10).Studies with pandemic influenza vaccines have also shown decreases indurability. In the AS03 avian influenza vaccine study, the GMT reached563 after 2 vaccine doses, but at 6 months post immunization, the GMThad dropped to 18, a 96% decrease (Leroux-Roels et al. (2010) Vaccine28:849). In the present tablet vaccine study, the percentage ofseroprotected subjects remained constant at 75% at 1 and 6 months postimmunization, and the HAI GMT titer drop was less dramatic showing onlya 28% decrease (FIG. 7B). One possibility is that the durability isbetter for vector-based vaccines because of enhanced T cell responses.

Example 5 Materials and Methods

Clinical protocol and enrollment. Subjects were pre-screened forHemagglutination Inhibition (HAI) titers within 45 days of enrollment.In order to be eligible for study participation subjects had to have aninitial HAI titer of ≤1:20, be between 18-49 years of age, and be ingood health. The active phase of the trial was through day 28, with thefollow-up phase for monitoring safety to continue for 1 year.

24 subjects were enrolled. All subjects that enrolled completed safetyand immunogenicity assessments through the active phase, and through day180 of the monitoring phase.

Randomization and Masking. The study was designed to evaluate thevaccine (VXA-A1·1) in 12 subjects at a single dose of 1×10¹¹ infectiousunits (IU) with 12 subjects given a placebo control. There were 3sequentially enrolled sentinel vaccine-treated subjects, with eachsubject dosed no more frequently than one every 24 h. After a week ofmonitoring for vaccine-related toxicities, the remaining subjects in thetreated cohort (9) were randomized along with 12 placebo controls.Randomization was performed by computer generated assignment, and studydrug was distributed with concealed identity to the blinded staff by theunblinded pharmacist. All investigative site staff as well as personsdirectly involved with immunological assays or the assessment ofclinical safety remained blind to treatment assignments. All subjectswere blinded in the study.

Vaccine. The rAd vector (non-replicating Ad5) carries DNA which encodesthe HA (A/CA/04/2009) transgene whose expression is driven by a CMVpromoter and a molecular dsRNA hairpin driven by a separate promoter.GMP drug substance was produced in Wave bags (GE Healthcare, Waukesha,Wis.) at Lonza Biologicals (Houston, Tex.). Purification was performedby ion exchange chromatography, followed by buffer exchange. Purifiedvector was mixed with excipients, lyophilized, and then tableted atLonza using microcrystalline cellulose and starch as tableting bulk.Tablets were enteric coated with Eudragit L100® (Evonik Industries,Darmstadt, Germany) using a Vector Hi-Coater system (Vector Freund,Cedar Rapids, Iowa). The final product was released in one lot, andtitered by standard IU assay at Lonza. Placebo was prepared as similarlysized and shaped tablets containing 150 mg of microcrystallinecellulose, without enteric coating.

Endpoints. The primary endpoint for this study is safety and thesecondary endpoint is immunogenicity through the active phase, primarilyby HAI titers and HAI seroconversions. Additional immunologicalendpoints include MN titers and ASCs. There were 5 adverse events in theplacebo group and 4 in the vaccine group, all of which were grade 1 inseverity. There were no serious adverse events reported in the study.

PBMC isolation and cryopreservation. Blood was collected in K₃ EDTAVacutainer® tubes (BD, Franklin Lakes, N.J.) and PBMCs were isolated thesame day using Lymphoprep™ tubes (Axis-Shield, Norway). PBMCs werefrozen and thawed using serum free reagents according to themanufacturer's instructions (Cellular Technology Ltd [CTL], ShakerHeights, Ohio).

Antibody Secreting cells (ASCs). Enzyme linked immunosorbent (ELISpot)kits for IgG and IgA secreting B cells were performed according tomanufacturer's instructions (Mabtech, Mariemont, Ohio). Cells werecultured (between 1·5×10⁴ to 5×10⁵ cells per well) in triplicate wells,in CTL-Test medium to optimize spots. HA protein (Protein Sciences Corp,Meriden, Conn.) was biotinylated and quantitated using a biotinylationkit (Pierce, Rockford, Ill.).

Antibody assays. HAI and Microneutralizing (MN) Titers were performedand were measured against MDCK derived A/CA/07/2009 and egg derivedA/CA/07/2009 respectively. HAI and MN titers less than 10 were marked as5 as suggested by regulatory advice.

Statistical analysis. Unpaired Students “t” tests were performed to testfor significant differences between groups. A two-tailed Fisher's Exacttest was used to determine if the observed frequencies were differentfor some analyses, as stated in the text. For both tests, p values of≤0.05 were considered significant. 95 percent confidence intervals (95CI) were provided for measured values.

Example 6 Nonclinical Studies of a Norovirus Vaccine. Introduction

The norovirus VP1 vaccines (VXA-G2.4-NS and VXA-G1.1-NN) of theinvention has the same replication-defective viral vector backbone andadjuvant RNA sequence as described in U.S. Pat. No. 8,222,224 andScallan et al. Clinical and Vaccine Immunology 2013; 20(1): 85-94. Thesequence of the vector backbone is provided in SEQ ID NO: 7.

VXA-G2.4-NS is an E1/E3-deleted replication-incompetent serotype 5adenovirus vector designed for use as a vaccine for the prevention ofnorovirus disease (NVD). The recombinant adenovirus (rAd) vector codesfor a 1.6 kb gene from the viral protein 1 (VP1) of norovirus (G2.4Sydney strain) and an adjuvant dsRNA sequence that enhances theimmunogenicity of the expressed antigen in the gut mucosa via its TLR3agonistic activity. The VP1 gene has been codon-optimized for expressionin mammalian cells and is expressed using a human cytomegalovirusintermediate early region (hCMVIE) enhancer/promoter and a bovine growthhormone polyadenylation (pA) signal. This expression cassette alsoincludes the first intron of human β-globin to enhance transgeneexpression. A second hCMVIE promoter is used to express the adjuvant RNAsequence. The adjuvant sequence is derived from a luciferase sequenceand has been reported to stimulate the induction of type I interferonsin vitro (2). The adjuvant is expressed as a short hairpin RNA,comprising a 21-nucleotide sequence (GAAACGATATGGGCTGAATAC) SEQ ID NO:11 as a tandem sequence in forward and reverse orientations separated bysix nucleotides that comprise the loop of the RNA. The 21-nucleotideforward and reverse RNA sequences anneal to form the stem of the loop.This adjuvant cassette utilizes a synthetic poly A (SPA).

As described below, preclinical studies in mice and ferrets show thatvaccination with VXA-G2.4-NS (and the related VXA-G1.1-NN) elicitedsubstantial and reliable antibody responses in both systemic serum IgGand intestinal (fecal) IgA response in test animals. A unique immuneresponse elicited by oral vaccination is the induction of antibodiesderived from local sources of B cells. After oral vaccination or entericinfection, B cells residing in the lamina propria (underneath the gutepithelial cells) predominantly produce dimeric antibodies of the IgAisotype . IgA antibodies pass across the gut epithelial cells into thelumen through facilitated transport, leaving a secretory componentattached to dimeric IgA. The resulting molecule is known as secretoryIgA or SIgA. SIgA serve as an additional external barrier to blockenteric infection. SIgA are eventually flushed out of the system, andcan be detected in fecal samples.

Nonclinical Pharmacology Introduction

The primary objective of the immunogenicity studies was to demonstratethat oral adenoviral constructs that express VP1 from two differentnorovirus species could elicit intestinal specific SIgA as well as serumIgG immune responses against norovirus antigen (VP1) following oralimmunization of norovirus vaccine.

The secondary objective of the immunogenicity studies was to compare tworoutes of immunization (oral with VXA-G2.4-NS vs. i.m. with VP1protein). The VP1 protein was compared since VP1 VLP based injectablevaccine is one of the few norovirus vaccines under development.Generally, SIgA are induced by mucosal immunization but a much lesserdegree by parenteral immunization. Therefore, we investigated intestinalSIgA response induced by oral VXA-G2.4-NS delivery compared toinjectable recombinant VP1 protein based vaccines.

TABLE 4 Nonclinical Pharmacology Studies Animal Endpoints ModelVXA-G2.4-NS (Sample Study Title (Species, Therapy (Immunizationcollection Endpoints (Study No.) Gender, No.) (Treatment) schedule) timepoints) (Assays) IMMUNOGENICITY STUDY Immunogenicity study ofAd-dsRNA-CMV-VP1 (Norwalk) vaccine Study No. (1) Balb/c mice. Group 1:oral Immunization on 8 weeks VP1 6 F per group VXA-G1.1-NN days 0 and 28ELISA (1 × 10⁸ IU^(a)) (serum IgG Group 2: oral and fecal VXA-G1.1-NNIgA) (5 × 10⁸ IU^(a)) (2) Balb/c mice. VXA-G1.1-NN: Immunization on 8weeks VP1 6 F per group oral days 0 and 28 ELISA (1 × 10⁹ IU^(a)) (serumIgG and fecal IgA) (3) Balb/c mice. Group 1: oral Immunization on 4 and8 VP1 6 F per group VXA-G1.1-NN days 0 and 28 weeks ELISA (1 × 10⁸IU^(a)) (serum IgG Group 2: i.m. and fecal VP1 protein from IgA) Norwalkvirus (1 ug) (4) Balb/c mice. Group 1: oral Immunization on 4 and 8 VP16 F per group VXA-G1.1-NN days 0 and 28 weeks ELISA (1 × 10⁸ IU^(a))(serum IgG Group 2: i.m. and fecal VP1 protein from IgA) Norwalk virus(1 ug) + Alum Immunogenicity study Balb/c mice. VXA-G2.4-NS:Immunization on 4 and 8 VPl ELISA of VXA-G2.4-NS 6 F per group oral days0 and 28 weeks Serum IgG (Sydney) vaccine (1 × 10⁸ IU^(a)) 8 weeks FecalIgA (5) Immunogenicity study Ferrets Group 1: oral Immunization on 8weeks ELISA of VXA-G2.4-NS 4M/4F per VXA-G2.4-NS days 0 and 28 (serumIgG (Sydney) vaccine group 1 and 2 (1 × 10⁹ IU^(a)) and fecal IgA) StudyGroup 2: oral Immunization on 8 weeks VP1 ELISA VXA-G2.4-NS day 0 and 2(serum IgG (1 × 10⁹ IU^(a)) and fecal IgA) 2M/2F per Group 3: i.m.Immunization on 8 weeks VP1 ELISA group 3 VP1 protein from days 0 and 28(serum IgG Sydney strain and fecal NoV (5 ug) IgA) ^(a)IU = infectiousunits

Immunogenicity Studies

The primary objective of the initial mice immunogenicity studies was todetermine if the vector backbone expressing a norovirus VP1 from Norwalkvirus or the Sydney strain could induce an antibody response to VP1 fromNorwalk virus or the Sydney strain as measured by ELISA.

rAd expressing the Norwalk VP2 strain and the dsRNA adjuvant(VXA-G1.1-NN) was delivered orally by gavage on days 0 and 28. A dosetitration was conducted (study#1 and study#2). Norwalk virus VP1specific IgG and sIgA responses were measured from serum and fecalsamples, respectively at 8 weeks. As expected, as the dose was increasedfrom 1×10⁸ to 5×10⁸ to 1×10⁹, the serum VP1 IgG titer showed adose-dependent increase geometric mean titer (GMT) increase from 2×10³to 1×10⁴ to 5×10⁵ (FIG. 8 ). A similar but slightly more gradual dosedependent increase from 1×10³ to 2×10³ to 3×10⁴ in fecal VP1 IgAresponse was also observed (FIG. 9 ). The mice studies showed thatVXA-G1.1-NN vaccine is immunogenic and generated dose dependent serumand fecal VP1 antibody responses.

In the next study (study#3), immune responses via two routes ofadministration with oral delivery by gavage with VXA-G1.1-NN (group 1)and intramuscular injection with Norwalk VP1 protein (group 2) werecompared (FIGS. 2.2 .3 and 2.2.4). Norwalk virus VP1 specific IgG andSIgA responses were measured from serum and fecal samples, respectivelyat 4 and 8 weeks. The oral vaccine of the invention generated slightlyhigher serum IgG titer values than the intramuscular protein vaccine(FIG. 10 ). In the fecal study, oral vaccine generated a dramaticallyhigher intestinal SIgA immune response than the intramuscular proteinvaccine (FIG. 11 ).

A similar study (study#4) was conducted with the Norwalk virus VP1protein vaccine with a vaccine adjuvant, aluminium hydroxide (FIGS. 12and 13 ). Intramuscular injection with the VP1 protein together withalum generated much higher serum titer (FIG. 12 ). However, the oralvaccine was clearly superior at generating SIgA, as indicated by thehigher fecal VP1 specific IgA titer value (FIG. 13 ). With the presenceof alum which is a strong adjuvant, a few animals produced fecal VP1 IgAafter intramuscular immunization whereas fecal VP1 IgA from sampleswithout alum was almost non detectable. The data suggest that the oralvaccine of the invention has an immunological advantage over injectablevaccine to produce intestinal SIgA immune response.

The currently circulating norovirus variant, the Sydney strain vaccine(VXA-G2.4-NS), was tested (study#5 FIG. 2.2 .7). At 4 weeks, the Sydneystrain vaccine generated far better titer values than the Norwalk virusvaccine. In addition, even at 4 weeks the titer value from the Sydneystrain vaccine was slightly higher than the Norwalk value at 8 weeks(FIG. 14A). The Sydney strain vaccine generated slightly higher fecalVP1 SIgA titer values than the Norwalk vaccine did (FIG. 14B). TheSydney strain vaccine appears to be more immunogenic. Accordingly, wechose the Sydney strain for the following ferret study.

The objective of the ferret study (study#6) was to (1) determine whetherthe oral vaccine of the invention could induce an intestinal SIgA aswell as systemic serum IgG response compared to recombinant Sydney VP1protein and (2) compare SIgA and IgG response with two differentimmunization schedules. Twenty ferrets were randomized into 3 groups (4males and 4 females in each of groups 1 and 2 and 2 males and 2 femalesin Group 3). On day 0 and 2, animals in group 1 were endoscopicallyadministered VXA-G2.4-NS. On days 0 and 28 animals in group 2 wereendoscopically administered VXA-G2.4-NS and animals in Group3 wereintramuscularly administered the Sydney norovirus recombinant VP1protein. Animals in group 1 produced higher IgG titer value than group 2(3×104 vs 5×103). Whereas animals in group 2 generated higher SIgA titervalues than group 1 (247 vs 92). Similarly to the mice study (FIG. 4 ),intramuscular VP1 protein vaccine failed to generate intestinal SIgAresponse although serum VP1 IgG response was well generated (8x104)(FIG. 15 ). Overall, the oral vaccine of the invention generatescomparable levels of serum IgG and superior levels of fecal IgA to VP1injectable protein vaccine.

Summary of Pharmacology Studies

The studies summarized within this section demonstrated that oraladministration of VXA-G2.4-NS could elicit substantial antibodyresponses to norovirus VP1 by ELISA in mice and ferrets. Local immunitycould play a very important role preventing infection and disease, andSIgA would likely be the most effective gut immune response to combat agut pathogen such as norovirus. These studies demonstrate thatVXA-G2.4-NS oral delivery generated intestinal SIgA response andsuperior levels of SIgA to VP1 injectable protein vaccine.

The duration of norovirus infection is relatively short. Norovirus hasaround 2 days of incubation time, followed by illness with vomiting anddiarrhea, which lasts 2 to 4 days. T and B cell responses start to occuraround 4 days after infection. Interestingly, the clearance of norovirusoccurs just after development of the early stage of T and B cells'activation. However, the oral vaccine of the invention could manipulatethe immune response to benefit the individual. Before norovirusinfection, the oral vaccine will induce gut homing B cells to secreteVP1 specific SIgA antibody. IgA enriched gut homing B cells in PBMC weredetected in previous studies. SIgA will pass across the intestinalepithelial cells to the alimentary tract (gut lumen). After norovirusinfection, SIgA will act as a blocking (neutralizing) antibody againstnorovirus infection. In conclusion, a gut pathogen is an excellent matchfor the oral platform because the oral delivery method activates immunecells with the desired gut homing property and intestinal SIgA is themost effective gut immune response to combat a gut pathogen.

Example 7 Phase 1 VXA-G24-NS Study Synopsis

This study is a phase 1, randomized, double-blind, placebo-controlled,dose-ranging trial to determine the safety and immunogenicity of anadenoviral-vector based norovirus vaccine (vxa-g2.4-ns) expressing gii.4vp1 and dsrna adjuvant administered orally to healthy volunteers. Thestudy will be conducted in 1-2 U.S. sites on healthy adult volunteersage 18 to 49 years. Subject participation in the study will last ˜1 yearfollowing successful screening and enrollment. After vaccinationsubjects will be followed for one month for efficacy (immunogenicity)and for 12 months for safety.

Investigational Product

VXA-G2.4-NS is an E1/E3-deleted replication-defective Adenovirusserotype 5 vaccine vector for prevention of noroviral gastroenteritiscaused by Norovirus GII.4. The vaccine vector encodes for a full-lengthVP1 (major capsid protein) gene from Norovirus GII.4 Sydney and isdescribed in detail in Example 6.

The GII.4 VP1 gene is expressed using a human Cytomegalovirus (CMV)intermediate early region (hCMVie) enhancer/promoter. In addition to thetransgene cassette, a second hCMVie promoter is used to express adouble-stranded RNA (dsRNA) sequence that acts as a TLR3-based adjuvant.

Control Product

A placebo tablet dosage formulation indistinguishable in appearance andnumber from the vaccine tablets

Regimen and Dosing

Single administration of VXA-G2.4-NS at a low dose of 1×1010 IU, a highdose of 1×1011 IU or placebo. The number of tablets per dose will becalculated based on the release assay results for the drug product. Twosentinel groups will enroll three subjects each in an open-label manner(Cohorts 1 and 3) to receive VXA-G2.4-NS prior to enrolling either ofthe randomized, controlled cohorts (Cohorts 2 and 4). Within thedouble-blinded groups (Cohorts 2 and 4), placebo subjects will receivethe same number of tablets as vaccine subjects. Subjects will beenrolled and dosed in the low dose group prior to initiation of dosingin the high dose group.

-   Cohort 1: 1×1010 IU±0.5 logs (n=3)-   Cohort 2: 1×1010 IU±0.5 logs (n=20) or placebo (n=10)-   Cohort 3: 1×1011 IU±0.5 logs (n=3)-   Cohort 4: 1×1011 IU±0.5 logs (n=20) or placebo (n=10)

Subjects in Cohorts 2 and 4 will be randomized in a 2:1 ratio toVXA-G2.4-NS at 1×1010 IU (low dose) or 1×1011 IU (high dose),respectively, or placebo.

Objectives

The primary objective is to determine the safety of a VXA-G2.4-NSnorovirus vaccine candidate. The secondary objective is to determine theimmunogenicity of a VXA-G2.4-NS norovirus vaccine candidate at two doselevels

Study Design

This is a Phase 1, randomized, placebo-controlled, double-blind (afterinitial open-label lead-in), dose-ranging study to assess the safety,reactogenicity and immunogenicity of an adenoviral-vector based oralNorovirus GII.4 vaccine and dsRNA adjuvant. All subjects will receive asingle vaccine administration.

The study will be enrolled in four cohorts:

-   Cohort 1: VXA-G2.4-NS at 1×1010 IU (low dose) sentinel-   Cohort 2: VXA-G2.4-NS at 1×1010 IU (low dose) or placebo-   Cohort 3: VXA-G2.4-NS at 1×1011 IU (high dose) sentinel-   Cohort 4: VXA-G2.4-NS at 1×1011 IU (high dose) or placebo

Cohort 1 (low dose sentinel group) will enroll 3 subjects to receive asingle dose of VXA-G2.4-NS at 1×1010 IU on Day 0. The 3 subjects will beenrolled sequentially (one per day), in an open-label manner. Uponcompletion of the Day 7 Visit in all 3 subjects, if no dose-limitedtoxicities are observed in these sentinel subjects (see Halting Rulesbelow), enrollment will begin in Cohort 2.

Cohort 2 (low dose randomized group) will randomize 30 subjects in a 2:1ratio to receive either VXA-G2.4-NS at 1×1010 IU (low dose) (n=20) orplacebo (n=10) in a double-blinded manner.

The study will enroll continuously during this phase unless the criteriafor pre-established stopping rules are met (see below). Should thishappen, enrollment of subsequent subjects will not be initiated untilthe study Safety Monitoring Committee (SMC) has completed review ofsafety data and offered the recommendation to proceed. The safetyassessment will be performed with the treatment assignments coded inCohort 2. If the SMC needs treatment information to assess an AE/SAE,the code will be revealed for that subject.

Upon completion of the Day 7 Visit in all 30 subjects, if nodose-limited toxicities are observed in these subjects (see HaltingRules below), enrollment will begin in Cohort 3. Cohort 3 (high dosesentinel group) will enroll 3 subjects to receive a single dose ofVXA-G2.4-NS at 1×1011 IU on Day 0. The 3 subjects will be enrolledsequentially (one per day), in an open-label manner. Upon completion ofthe Day 7 Visit in all 3 subjects, if no dose-limited toxicities areobserved in these sentinel subjects (see Halting Rules below),enrollment will in Cohort 4.

Cohort 4 (high dose randomized group) will randomize 30 subjects in a2:1 ratio to receive either a single dose of VXA-G2.4-NS at 1×1011 IU(high dose) (n=20) or placebo (n=10) in a double-blinded manner.

The study will enroll continuously during this phase unless the criteriafor pre-established stopping rules are met (see Halting Rules below).Should this happen, enrollment of subsequent subjects will not beinitiated until the SMC has completed review of safety data and offeredthe recommendation to proceed. The safety assessment will be performedwith the treatment assignments coded in Cohort 4. If the SMC needstreatment information to assess an AE/SAE, the code will be revealed forthat subject.

All subjects receiving study drug (vaccine or placebo) will have safetyand immunogenicity assessments completed for one month followingvaccination. Immunogenicity evaluations will be obtained at baselineprior to vaccination, and at Days 7 and 28 following vaccination.Subjects will also be evaluated for persistent immunogenicity at Day 180and be followed for safety for 12 months following vaccination.

Sample Size

The planned enrollment in this study is 66 subjects as follows:

-   Cohort 1: VXA-G2.4-NS (1×1010 IU±0.5 logs): n=3, sentinel-   Cohort 2: VXA-G2.4-NS (1×1010 IU±0.5 logs): n=20, or placebo: n=10;    total 30 subjects, 2:1 ratio-   Cohort 3: VXA-G2.4-NS (1×1011 IU±0.5 logs): n=3, sentinel-   Cohort 4: VXA-G2.4-NS (1×1011 IU±0.5 logs): n=20, or placebo: n=10;    total 30 subjects, 2:1 ratio

Additional subjects may be enrolled to replace dropouts.

Study Population

Male or female healthy volunteers, 18 to 49 years of age.

Inclusion/Exclusion Criteria

Inclusion criteria include:

-   1. Male or female volunteers aged 18-49 years, inclusive-   2. Able to give written informed consent-   3. Healthy (no clinically significant health concerns), as    determined by medical history, physical examination, 12-lead ECG,    and vital signs at screening-   4. Safety laboratory values within the following range criteria at    baseline:

a. Normal range for alkaline phosphatase (ALP), alanine aminotransferase(ALT), aspartate aminotransferase (AST), bilirubin, phosphorous(hypophosphatemia), neutrophils, occult blood, white blood cells (WBC),and urine protein;

b. Normal or grade 1 abnormality with no clinical significance (NCS) foralbumin, amylase, blood urea nitrogen (BUN), calcium, creatinephosphokinase (CPK), creatinine, glucose, magnesium, potassium, sodium,total protein, eosinophils (increase), hemoglobin, lymphocytes(decrease), and platelets;

c. Negative or positive with NCS for blood urine

-   5. Body mass index between 17 and 35 at screening-   6. Comprehension of the study requirements with ability and    willingness to complete all assessments and comply with scheduled    visits and contacts-   7. Female participants must have a negative pregnancy test at    baseline and fulfill one of the following criteria:

a. At least one year post-menopausal;

b. Surgically sterile;

c. Willing to use oral, implantable, transdermal or injectablecontraceptives for 30 days prior to and until 60 days after vaccination;

-   -   i. A reliable form of contraception must be approved by the        Investigator (e.g., double barrier method, Depo-Provera,        intrauterine device, Norplant, oral contraceptives,        contraceptive patches, abstinence)

Exclusion Criteria Include:

-   1. Receipt of any norovirus vaccine within two years prior to study    vaccination-   2. Administration of any investigational vaccine, drug or device    within 8 weeks preceding vaccination, or planned use of the above    stated during the study through the 12-month safety follow-up-   3. Administration of any licensed vaccine within 30 days prior to    vaccination-   4. Presence of significant uncontrolled medical or psychiatric    illness (acute or chronic) including institution of new    medical/surgical treatment or significant dose alteration for    uncontrolled symptoms or drug toxicity within 3 months of screening    and reconfirmed at baseline-   5. Any one of the following ECG findings within 30 days prior to    vaccination:-   a. QTc (Bazett) interval duration >450 msec (male) or >470 msec    (female),-   b. QRS interval greater than 120 msec,-   c. PR interval greater than 220 msec,-   d. Clinically significant ST-T wave changes or pathologic Q waves-   6. Positive serology for HIV-1 or HIV-2, or HBsAg or HCV antibodies-   7. Cancer, or treatment for cancer treatment, within past 3 years    (excluding basal cell carcinoma or squamous cell carcinoma)-   8. Presence of immunosuppression or medical condition possibly    associated with impaired immune responsiveness, including diabetes    mellitus-   9. Administration of any medications or treatments that may    adversely affect the immune system such as allergy injections,    immune globulin, interferon, immunomodulators, cytotoxic drugs or    other drugs known to be associated with significant major organ    toxicity, or systemic corticosteroids (oral or injectable) during 3    months prior to vaccination. Inhaled and topical corticosteroids    allowed-   10. Presence of household members who have received the Ad4 or Ad7    vaccines within 2 months prior to vaccination-   11. Presence of household members who are neonates, pregnant women,    or hematopoietic stem cell transplant or solid organ transplant    recipients-   12. History of drug, alcohol or chemical abuse within 1 year prior    to vaccination-   13. Receipt of blood or blood products 6 months prior to vaccination    or planned administration during the follow-up study period-   14. Donation of blood or blood products within 4 weeks prior to    vaccination or planned donation during the study period-   15. Acute disease within 72 hours prior to vaccination defined as    the presence of a moderate or severe illness with or without fever    (as determined by the Investigator through medical history and    physical examination)-   16. Presence of a fever ≥38° C. measured orally at baseline-   17. Stool sample with occult blood at screening-   18. Positive urine drug screen for drugs of abuse at screening-   19. Consistent/habitual smoking within 2 months prior to vaccination-   20. History of serious reactions to vaccination such as anaphylaxis,    respiratory problems, hives or abdominal pain-   21. Diagnosed bleeding disorder or significant bruising or bleeding    difficulties that could make blood draws problematic-   22. History of irritable bowel disease or other inflammatory    digestive or gastrointestinal condition that could affect the    distribution/safety evaluation of an orally administered vaccine    targeting the mucosa of the small intestine.    Such conditions may include but are not limited to:-   26. Any condition that, in the opinion of the Investigator, might    interfere with ability to assess the primary study objectives-   STUDY SCHEDULE The following study visits/calls will be conducted    during the study:

Screening Period (within 30 days prior to vaccination)

Day -2 (Baseline safety laboratory sample collection)

Day 0 Visit (Baseline; day of vaccination)

Day 2 Visit

Day 7 Visit

Day 28 Visit

Day 180 Visit

Day 365 End of Study Contact

Subjects will be followed via phone call daily on Days 1, 3 to 6, and14. They will also be contacted monthly between the Day 28 and Day 180Visits and also following the Day 180 Visit through Day 365 (end ofstudy). See Table 1 for detailed schedule of study procedures.

Safety and Immunogenicity Assessments Safety:

Safety and tolerability will be evaluated by: local (oral, esophagealand gastrointestinal) and systemic reactogenicity (solicited symptoms),and clinical and laboratory assessments. Physical exams, routineurinalysis, complete blood counts and serum chemistries will becollected pre-dose at Screening and Baseline (safety labs at Day −2) andat Study Days 2, 7 and 28. Vital signs will be recorded pre-dose atScreening and Baseline and at Study Days 2, 7, 28 and 180.

Safety will be evaluated using standard blood chemistry, hematology andurinalysis, and analyses performed per statistical methods below. Anysubject who experiences acute symptoms of conjunctivitis, upperrespiratory infection, loose stools and/or diarrhea within 14 daysfollowing initial vaccination, will be asked to return for a medicalevaluation and evaluation for adenoviral 5 infection. In these subjects,adenoviral cultures of throat and rectal swabs will be collected.

Immunogenicity:

Immunogenicity will be evaluated using cellular and humoral immunefunction assays from blood samples obtained at baseline (pre-dose) andat Study Days 7 and/or 28 depending on the assay. In addition, a finalevaluation for persistent immunogenicity will be performed at Day 180.The following assessments will be performed: Serum IgG VP1; Histo-bloodgroup antigen-blocking antibodies (BT50); IgG ASC VP1; IgA ASC VP1; Flowcytometric B cell immunophenotyping; Pre-plasma B cell culture for IgGVP1 and IgA VP1; Fecal IgA VP1; HAI; and Anti-Ad5.

Halting Rules

The study will be halted (no new enrollments and no furtherinvestigational product administered pending a full SMC safety review)if any of the below occur: For Cohorts 1 and 3 (3 subject open-labelsentinel lead in groups):

-   1. One or more subject experiences a vaccine-related SAE of any    grade;-   2. One or more subject experiences a Grade 3 or higher clinical AE    or laboratory abnormality;-   3. Two or more subjects experience any vaccine-related Grade 2    clinical AE or laboratory abnormality.

For Cohorts 2 and 4 (randomized, placebo-controlled groups):

-   1. One or more subject experiences a vaccine-related SAE of any    grade;-   2. Two or more subjects experience Grade 3 clinical or laboratory    AEs,-   3. One or more subject experiences a Grade 4 clinical AE, or a Grade    4 laboratory abnormality;-   4. Three or more subjects present with symptoms of adenovirus    infection and detectable replication competent adenovirus 5 vaccine    virus. If three or more subjects present with symptoms of adenovirus    infection, enrollment will be halted pending results for detection    of adenovirus 5 vaccine virus.

Endpoint Parameters

Safety analyses include: 1) Standard descriptive demography; 2)Proportion of subjects in each treatment group will be tabulated foreach local and systemic solicited reactogenicity event and anyunsolicited AEs. AE severity will be classified using standardizedcriteria adapted from the Sept. 2007 FDA Guidance entitled “ToxicityGrading Scale for Healthy Adult and Adolescent Volunteers Enrolled inPreventive Vaccine Clinical Trials”; 3) Subjects with AEs (includingclinical laboratory abnormalities) will be summarized by (1) MedDRA bodyorgan system and preferred term; (2) severity; (3) relatedness; and,separately, (4) seriousness; 4) Proportion of subjects in each treatmentgroup with AE reports within each body organ system will be compared inthe same manner. Significant heterogeneity will be probed at thepreferred term level

Immunogenicity analyses include: 1) Serum IgG VP1 and Histo-blood groupantigen-blocking antibodies (BT50); 2) Additional exploratory analyseswill include IgG ASC VP1; IgA ASC VP1; Flow cytometric B cellimmunophenotyping; Pre-plasma B cell culture for IgG VP1 and IgA VP1;Fecal IgA VP1; HAI; Anti-Ad5.

Statistical Methods Sample Size and Power:

This is the first in human clinical trial with VXA-G2.4-NS that will beconducted by the sponsor. There is currently no clinical informationabout the study drug. Hence the sample size was determined based onexperience of a typical Phase 1 vaccine study. The numbers of volunteersper group in Cohorts 2 and 4 are predicted to yield meaningfulimmunogenicity results. A sample size of 20 in vaccine group and 10 inplacebo group (i.e. 2:1 randomization ratio) will provide approximately86% power to detect a group difference, assuming the proportion ofresponse (observed in serum IgG VP1) in vaccine group is 50% and inplacebo is 0, using two-group Fisher's exact two-sided test atsignificance level of 0.05.

Data Analysis: Safety:

Safety will be summarized by treatment group. Local and systemicreactogenicity, AEs, clinical laboratory results, and vital signs willbe summarized descriptively by study visit. Number and percentage ofsubjects who experience acute symptoms of conjunctivitis, upperrespiratory infection, loose stools and/or diarrhea within 14 daysfollowing initial vaccination will be compared by treatment group usingFisher's exact test.

Immunogenicity:

Immunogenicity results evaluated by cellular and humoral immune functionassays from blood samples collected at preselected study visits will besummarized descriptively. Analysis of covariance (ANCOVA) will be usedin the analysis of the antibody titers, with post baseline log-titer asdependent variable, treatment as a factor, and baseline log-titer as acovariate. Least square (LS) means and 95% CI of the LS means will beobtained from the model. The post baseline Geometric (LS) Mean Titer(GMT) for the VXA-G2.4-NS group and Geometric (LS) Mean Fold Rise (GMFR)over the initial GMT at baseline will be reported.

Example 8 RSV Background

Respiratory syncytial virus (RSV) is the most important cause of lowerrespiratory tract infection (LRI) in infants and young children and is amajor cause of LRTI in the elderly and immune-compromised patients whereit can have devastating effects, causing significant morbidity andmortality. It is estimated to infect 5-10% of nursing home residents peryear, with rates of pneumonia or death of 10-20% and 2-5% respectively(Falsey et al. 2000). There is no approved vaccine though there is anapproved prophylactic monoclonal antibody, Palivizumab, for diseaseprevention in high-risk infants.

Given the lack of a vaccine Vaxart is addressing this large unmetmedical need by initiating a preclinical program to evaluate a RSVvaccine delivered using it's oral adenovirus vectored platform. Thisplatform has previously being used to deliver influenza vaccines topatients where it's efficacy and inducing significant immunity has beendemonstrated. An important strategy in this preclinical path isdemonstrating disease protection in a cotton rat RSV challenge model. Tothis end we have started such an evaluation and will have a completedata set shortly. We present preliminary data in this report, whichdemonstrates that significant immunity is generated even after a singlevaccination. Based on this immune response we expect the vaccine to beprotective.

RSVF Vaccine Evaluation in Cotton rats

A vaccine vector (Ad-RSVF) expressing the fusion protein (F) of RSV anda dsRNA adjuvant was generated as described above. The Ad vaccine vectorwas produced in 293 cells, purified and a titer determined. The vectorwas then evaluated first in mice where significant immune response waselicited to the fusion protein (data not shown) and then in the relevantRSV animal model, cotton rats. As an oral delivery method has not beenoptimized in Cotton rats the intra-nasal route was chosen as analternate mucosal route of delivery. Two doses were administered, a low(1e8 IU) and high dose (1e9 IU). Uninfected animals were used as anegative control and wild-type RSV virus (RSVA2) was used as a positivecontrol. Formalin inactivated RSV (FIRSV) was used as a control forundesired negative effects associated with an RSV vaccine developed inthe 1960's . After a single vaccination Ad-RSVF at both low and highdoses induced higher titers compared to wild-type RSV infection (FIG. 17) while the FIRSV induced a very weak immune response (200-300× lower).This data indicates that our vaccine is superior at inducing immunitythan WTRSV virus and more effective than the earlier FIRSVF vaccine.

In addition to total anti-Fusion protein antibodies (FIG. 17 ) we alsolooked at antibodies generated by our vaccine that could compete withthe approved Palivizumab antibody. In a competition assay using pooledsera from each of the treatment groups only the Ad-RSVF vaccinegenerated detectable antibody titers (FIG. 18 ). These data indicatethat the vaccine is producing RSV protective antibodies.

VXA-RSV-f Nonclinial Studies 8.1 Introduction

VXA-RSV-f is an E1/E3-deleted replication-incompetent serotype 5adenovirus vector designed for use as a vaccine for the prevention ofRSV and administered by the oral route. The recombinant adenovirus (rAd)vector codes for 1) the fusion (F) gene from the RSV A2 strain (Genbank#HQ317243.1) and 2) an adjuvant dsRNA sequence that enhances theimmunogenicity of the expressed F antigen in the gut mucosa via its TLR3agonistic activity. The vaccine backbone is identical to that inVaxart's ND1.1, VXA-A1.1 and VXA-BYW.10 vaccines for pandemic andseasonal influenza A and B, respectively, currently in clinicaldevelopment; the only change being that VXA-RSV-f has a differentsurface protein (F protein) being expressed. The RSV F protein gene hasbeen codon-optimized for expression in mammalian cells and is expressedusing a human cytomegalovirus intermediate early region (hCMVIE)enhancer/promoter and a bovine growth hormone polyadenylation (pA)signal. This expression cassette also includes the first intron of humanβ-globin to enhance transgene expression. A second hCMVIE promoter isused to express the adjuvant RNA sequence. The adjuvant sequence isderived from a luciferase sequence and has been reported to stimulatethe induction of type I interferons in vitro (1). The adjuvant isexpressed as a short hairpin RNA, comprising a 21-nucleotide sequence(GAAACGATATGGGCTGAATAC) SEQ ID NO: 11 as a tandem sequence in forwardand reverse orientations separated by six nucleotides that comprise theloop of the RNA. The 21-nucleotide forward and reverse RNA sequencesanneal to form the stem of the loop. This adjuvant cassette utilizes asynthetic poly A (SPA).

Vaxart has conducted preclinical studies to determine the immunogenicpotential of VXA-RSV-f in mice and cotton rats. These studies showedthat vaccination with VXA-RSV-f elicited substantial systemic serum IgGresponses in test animals.

As described above, Vaxart's RSV vaccine (VXA-RSV-f) will use the samereplication-defective viral vector backbone and adjuvant RNA sequence asthe company's pandemic and seasonal influenza virus programs. TheA/Indonesia/05/2005 (H5N1) pandemic influenza vaccine, ND1.1, ispresently being studied under BB-INDs 14660 and 15122, and theA/California/04/2009 (H1N1) seasonal influenza vaccine, VXA-A1.1, ispresently being studied under BB-INDs 15198 and 15285. TheB/Wisconsin/1/2010 (Yamagata) vaccine is presently being studied underBB-IND 16611. Because the only difference between the vaccines is theantigen gene [the F protein in VXA-RSV-f versus HA in VXA-A1.1, ND1.1and VXA-BYW.10], the preclinical studies from ND1.1 and VXA-A1.1 arerelevant to and support the clinical development of the VXA-RSV-fvaccine for prevention of RSV disease.

8.2 Nonclinical Pharmacology 8.2.1 Introduction

The primary objective of the immunogenicity studies of the RSV vaccinecandidate VXA-RSV-f was to demonstrate that vector construct couldelicit antibody responses in mice and cotton rats, and that the adaptiveimmune responses generated did not lead to enhanced RSV disease such asthat known to occur with the formalin inactivated RSV vaccine. Further,studies were performed in animals to demonstrate the value of theadjuvant for inducing antigen specific immune enhancement.

TABLE 5 Nonclinical Pharmacology Studies Endpoints Animal ModelVXA-RSV-f (Sample Study Title (Species, Therapy (Immunization collectionEndpoints (Study No.) Gender, No.) (Treatment) schedule) time points)(Assays) Immunogenicity Study of Ad-CMV-RSVf-dsRNA Vaccine Study No.Balb/c mice. Group 1: VXA-RSV-f Days 0 and 21 7 weeks RSV-F ELISA WCB2546 F per group i.n. (1 × 10⁸ IU^(a)) Group 2: VXA-RSV-f i.m. (1 × 10⁸ IU)Group 3: VXA-RSV-f oral (1 × 10⁸ IU) Study No. Cotton rats Group 1:VXA-RSV-f Days 0 and 28 8 weeks; RSV-F ELISA XV-95^(c) 6 F per group, ori.n. (1 × 10⁹ IU) Challenge day Day 5 post (serum IgG), anti- Cotton rat3 F per group for Group 2: VXA-RSV-f 56 challenge palivizumab challengeuninfected i.n. (1 × 10⁸ IU) competition assay, study control groupGroup 3: VXA-RSV-f PRNT antibodies. i.m. (1 × 10⁹ IU) RSV replicationGroup 4: rAd-Adj post challenge (1 × 10⁹ IU) Group 5:untreated/unifected Group 6: FI-RSV Group 7: Buffer alone Group 8:RSV/A2 1e5 PFU Study No. Cotton rats Group 1: Buffer alone Days 0 and 288 weeks; RSV-F ELISA XV-112^(c) 8 F per group, or Group 2^(b): oral VXA-Challenge day Day 5 post (serum IgG), anti- Cotton rat 6 F for bufferRSV-f (1 × 10¹⁰ IU) 56 challenge palivizumab immunogenicity controlgroup Group 3: oral VXA- competition assay, (and challenge) RSV-f (1 ×10⁹ IU) PRNT antibodies. Group 4: Oral VXA- RSV replication RSV-f (1 ×10⁸ IU) post challenge ^(a)IU = infectious units ^(b)oral delivery wasconducted by gavage by Sigmovir (Rockville, MD)

8.2.2 Immunogenicity and Challenge Studies

The primary objective of the initial mouse immunogenicity study (StudyNo. WCB254) was to determine if the Vaxart vector backbone expressingthe RSV F protein could induce an antibody response to RSV as measuredby ELISA. Following completion of the mouse study, two cotton ratstudies were performed. The objectives of the cotton rat studies were todemonstrate that VXA-RSV-f could elicit potent antibodies to RSV, andthat the vaccine could elicit protective immune responses against RSV.Further, the cotton rat studies were used to determine that VXA-RSV-finduced adaptive immune responses did not enhance RSV disease, such asthose recorded for the old formalin inactivated RSV vaccine (FI-RSV).

Immunogenicity Studies in Mice

Mice (6 females/group) were immunized with VXA-RSV-f using threedifferent routes of delivery in order to determine whether the constructwas immunogenic. Animals were immunized with 1×10⁸ IU on weeks 0 and 3using three different routes of delivery (intranasal, intramuscular, andoral). Antibody titers to RSV were measured on week 7. Results show thatboth the i.n. and i.m. routes of delivery were potent at elicitingantibody responses, and that oral administration was able to elicit someimmunity but was not as potent in mice as the other two routes ofdelivery. See FIG. 22 .

Immunogenicity and Challenge Studies in Cotton Rats

The objectives of the first cotton rat study (Study No. XV-95) were todetermine the ability of VXA-RSV-f to induce antibody responses and toprotect against RSV disease and viral replication. The cotton rat isconsidered an important model for preclinical development of RSVvaccines because of the susceptibility of the animal to RSV infection,and the reproducibility of the lung inflammation/cytokine skewingphenotype when given formalin inactivated RSV vaccine (FI-RSV) followedby RSV challenge (2). Female cotton rats (N=6 per group) were immunizedwith VXA-RSV-f by intranasal (i.n.) and intramuscular (i.m.) delivery onweeks 0 and 4 at 1×10⁹ IU. A lower dose i.n. delivery group at 1×10⁸ IUof VXA-RSV-f (VXA-RSV-f low) was also used. The VXA-RSV-f vaccinetreated animals were compared for ELISA IgG titers on week 8 with a noinfection/no vaccine control group (No infection), an adenovirus storagebuffer alone (buffer) group and a FI-RSV vaccine group at 1:100 dilutionof a stock (Lot #100) made by Pfizer from the RSV-A2 strain (3). To showthat post challenge results were antigen specific, a rAd that expressesthe adjuvant without the antigen at 1×10⁹ IU (Ad-Adj) was given i.n. andused as a control. As a positive control for immunogenicity and adaptiveimmune mediated protection, a single dose of wild-type RSV strain A2(RSV2) at 1×10⁵ PFU was given on week 0. Refer to Table 5 for summary ofstudy treatment groups, time points and endpoints.

All VXA-RSV-f immunized animals induced significant IgG antibody titersto RSV, with group average antibody titers exceeding 1×10⁵ (FIG. 23A) atweek 8. The FI-RSV and the RSV2 immunized animals also inducedsignificant IgG antibody titers, with average titers reaching 1×10⁴ onweek 8 (FIG. 23A). The VXA-RSV-f vaccine treated animals were also ableto induce antibodies that competed for the palivizumab binding site onthe F protein of the RSV virus, as measured by the competitive bindingELISA, whereas the FI-RSV vaccine did not induce antibodies that wereable to recognize the palivizumab binding site (FIG. 23B). There wassome ability of the wild-type RSV2 group (positive control) to induceantibodies that compete for the palivizumab binding site. Neutralizingantibodies to RSV were measured by PRNT assay. A similar trend to thepalivizumab analysis was seen, with all VXA-RSV-f groups able to inducesignificant neutralizing antibodies to RSV, whereas the FI-RSV vaccinewas not able to induce substantial neutralizing titers (FIG. 23C). RSV2control vaccine was able to elicit similar neutralizing titers as theVXA-RSV-f groups statistically speaking, with the VXA-RSV-f (i.m.)having the highest geometric mean titer (p=0.47 by Mann-Whitney).

The objectives of the second cotton rat study (Study No. XV-112) were todetermine if cotton rats could be immunized orally with the VXA-RSV-fvaccine and induce significant immune responses. Challenge was optional,subsequent to effective oral immunization. Oral immunization can bedifficult in animals, and prior documented experience for rAd oraldosing in cotton rats was not available. For this reason, a dosetitration of the VXA-RSV-f vaccine was performed with doses at 1×10⁸,1×10⁹, and 1×10¹⁰ IU given by oral gavage to stomach-neutralized cottonrats to approximate human oral tablet delivery. Animals were immunizedon weeks 0 and 4 and the antibody titers were measured on weeks 4 and 8.A buffer only group was used as a control, to show the backgroundeffects of no immunization.

The results show that both 1×10⁹ and 1×10¹⁰ doses could inducesignificant antibody titers to RSV-F on week 4, with significantboosting at week 8 (FIG. 24A). The 1×10⁸ IU vaccine group trended lowerin terms of total IgG antibody responses to RSV. Measurement of theability of oral VXA-RSV-f to induce antibodies that could compete forthe palivizumab binding site showed a dose dependent effect with higherdoses showing higher average competitive titers than the lower 1×10⁸ IUdose (FIG. 24B). Induction of neutralizing antibodies to RSV increasedwith higher vaccine doses, with the 1×10⁹ and 1×10¹⁰ IU VXA-RSV-fvaccine doses eliciting the higher neutralizing titers (FIG. 24C).

Following immunization in the first cotton rat experiment (XV-95, FIG.23 ), cotton rats were given wild-type RSV strain A2 at 1×10⁵ PFU at day56. Lungs and nasal passages were harvested 5 days later and analyzedfor the ability of the vaccines to protect against RSV replication anddisease in cotton rat tissue. Immunization with the VXA-RSV-f vaccineprovided complete protection against RSV replication in both lungs andthe nose, whereas the formalin inactivated vaccine (FI-RSV) and adjuvantalone (Ad-Adj) groups provided no protection against RSV replication inthe nose and only limited protection in the lungs with the FI-RSVvaccine (FIG. 25A). Maximal replication (as seen in the buffer controlgroup) had an average of 4.9 log₁₀ PFU RSV/g of lung tissue, and vaccineinduced immunity was able to reduce the post challenge RSV titers belowthe detectable level, a greater than 3 log₁₀ decrease.

Lung inflammation was measured by immunohistology and qRT-PCR analysison day 5 post RSV infection. Four different regions were assessed byimmunohistology to determine if the vaccine led to adaptive immuneenhancement of disease. The VXA-RSV-f vaccine did not inducesignificantly increased lung pathology scores for peribroncolitis (PB),perivasculitis (PV), interstitial pneumonia (IP), and alveolitis (A) ascompared to the FI-RSV vaccine, the “positive” control for lunginflammation (FIG. 25B), and did not lead to increases in the relativeabundance of IL-4 or IL-13 (FIG. 25C). Groups given i.n. administrationtrended higher for PB and PV, including the Buffer, RSV2, and adjuvantcontrol groups, but not necessarily for IP and A (FIG. 25B). Theadjuvant group (without expression of RSV F protein) induced a highlevel of PB post RSV challenge, but induced only low levels of IP and A.Buffer and adjuvant alone control groups did not induce a significantincrease in the relative abundance of IL-4 or IL-13 mRNA as the FI-RSVgroup did (FIG. 25B). The FI-RSV vaccine group induced an averagerelative abundance above 1 for IL-4 and above 3 for IL-13, compared tobelow 0.1 and 0.3 respectively for the VXA-RSV-f and RSV2 vaccine groups(FIG. 25C).

The oral cotton rat immunogenicity study (Study No. XV-112) waschallenged with wild-type RSV because of the potent neutralizingantibody titers observed. Following immunization, cotton rats were givenwild-type RSV strain A2 at 1×10⁵ PFU at day 56. Lungs and nasal passageswere harvested 5 days later and analyzed for the ability of the vaccinesto protect against RSV replication and disease in cotton rat tissue.Oral immunization with the VXA-RSV-f vaccine induced dose dependentprotection against RSV replication in both lungs and the nose, with thehighest dose vaccine inducing complete protection in the lungs andnearly complete protection in the nose; 8 out of 8 animals were negativefor RSV titers in the lungs and 7 of 8 animals were negative for RSVreplication in the nose (FIG. 26A). Maximal replication (as seen in thebuffer control group) had on average of 5.2log₁₀ PFU RSV/g of lungtissue, and vaccine induced immunity was able to reduce the postchallenge RSV titers greater than 3 log₁₀ for the 1×10⁹ and 1×10¹⁰ IUvaccine groups (FIG. 26A). The lowest dose group (1×10⁸ IU) alsodemonstrated substantial protection, with 6 of 8 animals having completeprotection. Inflammation was assessed as before. The vaccine groupstrended higher for inflammation compared to the buffer control group,but were not statistically different (FIG. 26B). Cytokine analysis ofthe lungs post challenge saw no substantial increase in the relativeabundance of the IL-4 or IL-13 mRNA as measured by qRT-PCR. All vaccineand the buffer control groups had relative abundance levels of IL-4 andIL-13 mRNA below 0.04 and 0.005, respectively (FIG. 26C). These valuesare extremely low compared to the relative abundance levels of 1 and 3for IL-4 and IL-13 respectively induced by the FI-RSV group in Study No.XV-95 (FIG. 25C), suggesting that no meaningful induction of thesecytokines has occurred following oral delivery of VXA-RSV-f.

Example 9 High Titer Neutralizing Antibodies to Influenza Following OralTablet Immunization: A Randomized, Placebo-Controlled Trial Introduction

Seasonal influenza vaccination requires substantial yearly campaigns tocollect enough fertilized eggs and massive machinery to harvest andprocess each mini-egg bioreactor. Cell culture or plant derivedhemagglutinin (HA) may reduce the burden of egg acquisition andprocessing, but these approaches still require expensive sterile filland finish to produce individual syringe needles, that need to bedisposed of as a biohazard. During a pandemic, schools can be closed andsocial distancing mandated, yet mass influenza immunization campaignstypically require lining up subjects at health clinics for injections.In order to circumvent this dilemma, oral influenza vaccines could besent through the mail thus avoiding most human to human contact. Maildelivery is already used for a wide-variety of oral medications and hasalready been suggested as a means to deliver critical medicines toveterans during a pandemic. Further, tableting is a rapid, sanitaryprocess that does not require the expensive sterile fill and finishingprocess that injected vaccines require.

Several adenoviral vector approaches have been attempted to enable oralinfluenza immunization. In 2011, a clinical trial using a cell-cultureproduced oral adenovirus vectored avian influenza (H5) vaccine wasinitiated. T cell responses to influenza H5 HA were measured in greaterthan 75% of subjects, but no neutralizing antibody responses wereobserved (Peters et al. Vaccine 2013; 31: 1752-8). After additionalformulation development and dose optimization was performed, theclinical trial that is the subject of this report was initiated forseasonal influenza, using a tablet delivery format instead of a capsule.The same vector backbone was used as before, but with a new HA sequencefrom a strain similar to the H1N1 strain of the current commercialseasonal influenza vaccine (A/California/04/2009 (H1N1)) and with a 10fold higher dose. A single dose of rAd-HA(A/CA/04/2009)-dsRNA was testedfor safety and immunogenicity, in a double-blind, randomized, controlledclinical study. This report summarizes the findings of this trial.

Materials and Methods Clinical Protocol and Enrollment

This was a phase 1, sequentially enrolled study, with a randomized andplacebo-controlled cohort to evaluate safety, and immunogenicity of arecombinant Ad serotype 5 (rAd5) based oral vaccine against H1 seasonalinfluenza, The study was conducted in accordance with applicable GoodClinical Practice guidelines, the United States Code of FederalRegulations, and the International Conference on Harmonizationguidelines. IRB approval was obtained from Aspire IRB (Santee,California; AAHRPP accredited) before enrollment of subjects. Studyparticipants were recruited using the CRO/Phase 1 Unit's existingvolunteer database as well as using IRB approved advertising (print ads,radio ads and social media). Informed consent was obtained from allsubjects after discussion of the study procedures and potential risks.

Subjects were pre-screened for Hemagglutination Inhibition (HAI) titerswithin 45 days of enrollment. In order to be eligible for studyparticipation subjects had to have an initial HAI titer of <1:20, bebetween 18-49 years of age, and be in good health. Additional enrollmentcriteria are listed at clinicaltrials.gov under NCT01688297. The activephase of the trial was through day 28, with the follow-up phase formonitoring safety to continue for 1 year.

Randomization and Masking

The study was designed to evaluate the vaccine (VXA-A1·1) in 12 subjectsat a single dose of 1×1011 infectious units (IU) with 12 subjects givena placebo control. There were 3 sequentially enrolled sentinelvaccine-treated subjects, with each subject dosed no more frequentlythan one every 24 h. After a week of monitoring for vaccine-relatedtoxicities, the remaining subjects in the treated cohort (9) wererandomized along with 12 placebo controls. Randomization was performedby computer generated assignment, and study drug was distributed withconcealed identity to the blinded staff by the unblinded pharmacist. Allinvestigative site staff as well as persons directly involved withimmunological assays or the assessment of clinical safety remained blindto treatment assignments. All subjects were blinded in the study.

Sample Size

The overall number of volunteers per test group (n=12) was predicted toyield meaningful results. This was defined in a prior study as observing50% of responders in the vaccine group and none in placebo group. Withthe sample size of 12 per group, there is 80% power to detect a groupdifference, assuming the proportion of response (defined as HAI>40) invaccine group is 50% and in placebo is 0, using two-group Fisher's exacttwo-sided test at significance level of 0.05.

Vaccine

The rAd vector (non-replicating Ad5) carries DNA which encodes the HA(A/CA/04/2009) transgene whose expression is driven by a CMV promoterand a molecular dsRNA hairpin driven by a separate promoter, asdescribed before (Scallan et al. Clinical and Vaccine Immunology 2013;20(1): 85-94). GMP drug substance was produced in Wave DisposableBioreactors (GE Healthcare, Waukesha, Wis.) at Lonza Biologicals(Houston, Tex.). Purification was performed by ion exchangechromatography, followed by buffer exchange. Purified vector was mixedwith excipients, lyophilized, and then tableted at Lonza usingmicrocrystalline cellulose and starch as tableting bulk. Tablets wereenteric coated with Eudragit L100® (Evonik Industries, Darmstadt,Germany) using a Vector Hi-Coater system (Vector Freund, Cedar Rapids,Iowa). The final product was released in one lot, and titered bystandard IU assay at Lonza. Placebo was prepared as similarly sized andshaped tablets containing 150 mg of microcrystalline cellulose, withoutenteric coating.

Safety Assessments

The principal investigator (PI) assessed solicited and unsolicitedadverse events (AEs) in a blinded manner. The SMC oversaw the safety ofthe study but did not participate in the grading of AEs. Solicited AEs(reactogenicity) were collected with the aid of a 7-day solicitedsymptoms diary card. Unsolicited AEs (all other clinical AEs) werecollected with the aid of an unsolicited diary card through Day 28.Investigators used the Center for Biologics Evaluation and Research(CBER) Guidance for Industry: Toxicity Grading Scale for Healthy Adultand Adolescent Volunteers Enrolled in Preventive Vaccine Trials,September 2007 to grade AEs.

Because of the novel adjuvant, we collected data on the occurrence ofAEs of special interest (AESIs) and new onset of chronic illnesses(NOCIs). These include neuroinflammatory disorders, musculoskeletaldisorders, gastrointestinal disorders, metabolic diseases, skindisorders and other autoimmune disorders. No AESIs or NOCIs have beenreported through day 180 following immunization.

Endpoints

The primary endpoint for this study is safety and the secondary endpointis immunogenicity through the active phase, primarily by HAI titers andHAI seroconversions. Additional immunological endpoints include MNtiters and ASCs.

PBMC Isolation and Cryopreservation

Blood was collected in K3 EDTA Vacutainer® tubes (BD, Franklin Lakes,N.J.) and PBMCs were isolated the same day using Lymphoprep™ tubes(Axis-Shield, Norway).

PBMCs were frozen and thawed using serum free reagents according to themanufacturers instructions (Cellular Technology Ltd [CTL], ShakerHeights, Ohio).

Antibody Secreting Cells (ASCs)

Enzyme linked immunosorbent (ELISpot) kits for IgG and IgA secreting Bcells were performed according to manufacturer's instructions (Mabtech,Mariemont, OH). Cells were cultured (between 1.5×104 to 5×105 cells perwell) in triplicate wells, in CTL-Test medium to optimize spots. HAprotein (Protein Sciences Corp, Meriden, Conn.) was biotinylated andquantitated at Vaxart using a biotinylation kit (Pierce, Rockford,Ill.). Spots were counted at Zellnet Consulting Inc (Fort Lee, N.J.).

Antibody Assays

HAI and MN Titers were performed by Focus Diagnostics (Cypress, Calif.)similarly as described previously (Greenberg et al. The New EnglandJournal of Medicine 2009; 361(25): 2405-13). HAI and MN were measuredagainst MDCK derived A/CA/07/2009 and egg derived A/CA/07/2009respectively. HAI and MN titers less than 10 were marked as 5 assuggested by regulatory advice. Adenovirus neutralizing titers weremeasured as described before 2.

Statistical Analysis

In general, descriptive statistics for continuous variables included thenumber of subjects with data to be summarized (n), mean, standard error(std err), and 95% confidence intervals (95 CI). Titers were reportedwith geometric means and 95 CI. Categorical variables were presentedusing frequency counts and percentages. Treatment group differences werecompared using two-group t-test in continuous variables and Fisher'sExact test in categorical variables. All statistical tests weretwo-sided at a significance level of 0.05 without adjusting formultiplicity. An analysis of covariance (ANCOVA) model was used for (logtransformed) HAI antibody titers, with Day 28 log-titer as dependentvariable, treatment as a factor, and Day 0 log-titer as a covariate.Least square (LS) means, 95 CI of the LS means, difference of LS meansand the 95 CI of the difference of LS means were obtained from themodel. As an exploratory analysis for the HAI antibody titer, anotherANCOVA model included age, sex, and body mass index (BMI) as covariateswas performed as well.

Results Demographics

365 subjects were screened and 24 subjects were enrolled. All subjectsthat enrolled completed safety and immunogenicity assessments throughthe active phase, and through day 180 of the monitoring phase (FIG. 19). Demographics are described in Table 8 for both placebo and vaccinetreated subjects.

Summary of Adverse Events

In the first 7 days following test article administration, there were 8total solicited adverse events (AEs) reported in the VXA-A1·1 vaccineand placebo groups (Table 9). All of these AEs were grade 1 in severity.The investigator's assessment as to whether the AE was related totreatment is also indicated (Table 9). The most frequent AE was headache(2 in placebo, and 1 in the vaccine group). All other solicited AEs weresingle events (Table 9). There were a total of 8 unsolicited AEs invaccine and placebo groups in the 28 days following immunization, with 3events occurring in the placebo and 5 events occurring in the vaccinegroup. There were no serious adverse events reported in the study.

Clinical laboratory abnormalities were distributed across the vaccineand placebo groups. Of note, there were 6 neutropenic events in thevaccine group and 4 in the placebo group. These events occurred in atotal of 8 subjects, 4 of who had pre-treatment neutropenic bloodcounts. Five of these subjects were also black or Japanese, which areethnic groups that have a relatively high frequency of benign ethnicneutropenia (BEN). As is the case with (BEN), there were no clinicalmanifestations that resulted from any of the neutropenic eventsreported.

Immunogenicity Results

HAI responses were measured on days 0 and 28 (FIG. 20A). No placebotreated subject seroconverted, but one placebo had a high day 0 value(which would have excluded the subject if measured at screening). Noneof the vaccine subjects had a starting HAI titer >20. Afterimmunization, nine subjects in the vaccine group reached seroprotectivelevels (HAI ≥40) (FIG. 20A). Of the eleven 4-fold risers (92%), nineseroconverted (SC) with the other 2 subjects showing a 4-fold increasein HAI titer from 5 to 20. The vaccine group had a statisticallysignificant increase in the number of 4-fold responders versus placebo(11 versus 0, with P<0·0000 by Fisher's Exact Test). Using an ANCOVAmodel accounting for the Day 0 log-titer as a covariant, the Geometric(LS) Mean Titer (GMT) for the vaccine group was calculated to be 71·5(95 CI: 45-114) on Day 28, a 7·7-fold Geometric (LS) Mean Fold Rise(GMFR) over the initial GMT of 7·9 (95 CI: 4·6-13·6) on Day 0. The GMTon Day 28 for placebo group was 10·1 (95 CI: 6·4-16·2) on Day 28, a1·1-fold GMFR over initial GMT of 11·0 (95 CI: 6·4-18·9) on Day 0.Comparing to placebo, the vaccine group had a statistically significantincrease in GMT on Day 28 (p-value <0.001). The covariate effect ofbaseline was also statistically significant (p-value <0.001). Anexploratory analysis was also carried out using another ANCOVA model,where additional covariates, age, sex and BMI were included. The effectsof these covariates were not statistically significant on Day 28[p-values: 0.993 (for age), 0.696 (for sex), 0.201 (for BMI)].

Durability of the antibody response was measured by examining HAIresponses 180 days after immunization. In the vaccine-immunized group,75% (9 of 12) of the subjects were seroprotected on day 28 and 75% (9 of12) were still seroprotected on day 180. The HAI GMT were plotted (FIG.20B), and the decrease in the GMT was found to be 29% between 28 and 180days post immunization.

Neutralizing antibody responses to influenza were measured by MN assay.Significant increases in the MN titers in the treated group versus theplacebo control were observed (FIG. 20C). The frequency of 4-fold MNresponders in the vaccine treated group was significantly different thanthe placebo group, with 11 subjects responding in the vaccine treatedgroup versus 0 in the placebo group (P<0·0000 by Fisher's exact test).

After removing subjects that had baseline MN titers (and HAI titers)greater than 40, the geometric mean titers (GMT) were calculated in theremaining subjects on days 0 and 28 (Table 10). The GMT for the vaccinegroup rose to 247 (95 CI: 89-685) versus no rise in the placebo for aday 28 GMT of 9.6 (95 CI: 5-18). These calculations had no impact on thevaccine group, as none of the subjects had high initial MN or HAItiters. These results show that neutralizing antibody titers toinfluenza are generated by oral immunization, with a greater than20-fold increase in the GMT after immunization in the vaccine-treatedgroup.

In order to measure total antibody responses to HA, the numbers ofcirculating pre-plasma B cells in peripheral blood were measured by ASCassay on days 0 and 7 after immunization. Results show that ASCs couldbe reliably measured on day 7 in the vaccine-treated group (FIG. 20D).Background ASCs were generally negligible on day 0. For the vaccinetreated group, an average of 992 (+/−std err 209, 95 CI: 532-1452) IgGASCs and 337 IgA ASCs (+/−std err 104, 95 CI: 117-580) each per 1×106PBMC were found for day 7, with only one subject out of 12 having nodetectable ASC response. The placebo group had no IgA spots on day 7,but one subject had a high background smear and a measurable IgG ASCresponse with smaller spots than normally observed. The treated groupwas significantly different than placebo in terms of the ability toelicit an IgG or an IgA ASC response at day 7 (P=0·0007 and P=0.008respectively by T Test).

Subjects were measured for their anti-vector titers pre- andpost-immunization. Following oral immunization, a few vaccine-treatedsubjects had an increase in neutralizing antibody responses to Ad5,which led to a 2·6-fold increase in the GM neutralizing antibody titers,compared to 1·0-fold GM fold rise in the placebo treated subjects. Inthe vaccine group, HAI and MN responses trended similarly for individualsubjects. Eight subjects were Ad5 negative before immunization, and fourwere Ad5 positive before immunization. One subject that was Ad5 positivedid not HAI seroconvert, however, one subject that was Ad5 positive hadthe highest increase in HAI titers (64 fold) of any of the subjects inthe study (FIG. 21B). This same subject had a gain in MN titers of 362fold (FIG. 21A) without any increase in the Ad5 neutralizing antibodytiters pre and post immunization. There was no observed correlationbetween starting Ad5 titers versus fold MN response (or HAI response)for the subjects immunized with the tablet vaccine (FIG. 21A and 21B).

Discussion

When the US military conducted an independent study to measure theeffects of their seasonal vaccine campaigns on neutralizing antibodyresponses in military personnel, they reported a MN Titer GMFR of 5·6after trivalent inactivated vaccine (TIV) injection and a GMFR of 2·2following live-attenuated influenza vaccine (LAIV) intranasaladministration after accounting for subjects that had MN titers above 40to start (Faix et al. PloS one 2012; 7(4): e34581). In this study, theMN GMFR was calculated at 29 for the 12 vaccine treated subjects (Table10) with 92% of subjects showing a greater than 4-fold rise in MNtiters. In the study by Gordon, et al, the SC rate to H1N1 was found tobe 45% for one injection of 45 ug of HA protein (without adjuvant)(Gordon et al. Vaccine 2012; 30(36): 5407-16). This contrasts with theresults published by Greenberg, et al, (supra) where the H1N1 vaccinewas highly immunogenic and a 78% SC rate was observed after 1 dose of asplit vaccine. In our tablet study, the HAI SC rate among vaccinetreated subjects was 75% with over 92% of subjects having a 4-fold risein HAI titers (FIG. 20A). It is not clear why our MN titers are muchhigher than the HAI titers. It is possible that the MN assay is justmore sensitive or that the oral rAd based vaccine elicits strongerneutralizing responses outside the head region than protein injectedvaccines. In either case, our early stage results suggest that an oraltablet vaccine would be competitive with existing vaccines in terms ofeliciting neutralizing antibody responses to influenza.

Individuals with pre-existing immunity to influenza H1N1 were excludedfrom study participation. This was helpful in analysis of immuneresponses in this Phase 1 study to better understand effects of thevaccine. In practice, in the “real world”, individuals with and withoutpre-existing immunity to influenza are immunized. Enrollment in Phase 2and 3 studies will include individuals with and without detectableantibody levels to vaccine antigen at baseline.

HAI responses are elicited with injected commercial vaccines, but HAItiters are known to wane. In a study by Crum-Cianflone, et al, non-HIVinfected volunteers had a 67% drop in GMT HAI titers between 1 and 6months post immunization (Crum-Cianflone et al. Vaccine 2011; 29(17):3183-91). Similarly, the percentage of seroprotected subjects droppedfrom 75% to 56% for HIV negative subjects that enrolled withseronegative HAI titers (≤1:10)10. Studies with pandemic influenzavaccines have also shown decreases in durability. In the AS03 avianinfluenza vaccine study by Leroux-Roels, et al, the GMT reached 563after 2 vaccine doses, but at 6 months post immunization, the GMT haddropped to 18, a 96% decrease (Leroux-Roels et al. Vaccine 2010; 28(3):849-57). In our tablet vaccine study that enrolled seronegative subjects(all subjects ≤1:20), the percentage of seroprotected subjects remainedconstant at 75% at 1 and 6 months post immunization, and the HAI GMTtiter drop was less dramatic showing only a 29% decrease (FIG. 20B).While unproven, one possibility is that the durability is better forvector-based vaccines because of enhanced T cell responses. Thesepreliminary data are at least encouraging that the tablet vaccine canprovide antibody durability.

The numbers of clinical reported adverse events were similar to thoseobserved before for other adenoviral vectored based vaccines of theinvention. In this study, 17 clinical adverse events were reportedthough day 28 following immunization and are reasonably evenlydistributed between placebo and treated groups. In a published study ofan recombinant adenovirus EBOV vaccine injected into humans, thefrequency of any adverse event was 55% among vaccine recipients and 25%among placebo recipients, with the most common reported adverse eventbeing headaches (55%), myalgia (46%), and chills (27%) in the high dosevaccine group (Ledgerwood et al. Vaccine 2010; 29(2): 304-13). The mostfrequently reported adverse event in our vaccine tablet study washeadache (reported in 1 vaccine and 2 placebo subjects).

Although Ad5 immunity can be an issue with injected vaccines, it may notbe the case with oral immunization with a non-replicating vector whereneutralizing antibody titers do not seem to hinder performance (Xiang etal. Journal of virology 2003; 77(20): 10780-9). The ability to elicit aneutralizing immune response to influenza did not appear to be impactedafter oral immunization with the vaccine tablet (FIG. 21 ). One of thesubjects with the highest anti-Ad5 titer to start had the highestmeasured increase in neutralizing antibody responses by MN and HAI assay(FIG. 21 ). While i.m. immunization can cause 100% Ad5 seroconversionand GMTs to rise greater than 50 fold (estimated from FIG. 2 b fromO′Brien et al. Nat Med 2009; 15(8): 873-5), the increase in neutralizingtiters with the 1×1011 IU tablet were much more modest. Oralimmunization appears to lead to a much more selective increase in theimmune response to transgene compared to vector with a GMT MN titer riseof 29 compared to Ad5 titer increase of 2.6. In contrast to results withnon-replicating Ad5 based vaccine in humans, oral administration ofreplicating vectors shows the opposite trend where the antibodyresponses to the vector far exceeds the antibody responses to transgene.As an example, in an oral replicating Ad4-HA study, 80% of subjectsseroconverted to Ad4 on the first immunization, but did not havesignificant neutralizing antibody responses to HA after 3 immunizations(with a maximum of 19% SC in a vaccine group) (Gurwith et al. The Lancetinfectious diseases 2013; 13(3): 238-50).

An oral formulation could greatly facilitate vaccine administration,particularly during a pandemic when rapid distribution is needed. Duringthe 2009 H1N1 pandemic, when vaccine was in short supply, individualcounty health departments in California had to have a plan fordistribution. In Los Angeles County, approximately 60 points ofdistribution (PODs) were tasked to administer the vaccine. Approximately247 persons per hour lined up, and the rate of immunization wasapproximately 239 person per hour at each POD (Saha et al. Emerginginfectious diseases 2014; 20(4): 590-5). For the PODs in Los AngelesCounty, this translated to 143,000 people per day. In a city of 9million, assuming supplies or qualified personnel were not in shortsupply, it would take more than 60 days to complete an immunizationcampaign. As an alternative, if the H1N1 pandemic vaccine was deliveredby US mail and self-administered by tablet, all 9 million subjects couldbe immunized within a single day without people having to stand in line,and risking exposure to the growing outbreak. While there are regulatoryhurdles to overcome, our tablet vaccine appears to be stable at roomtemperature for greater than 270 days and can tolerate short-termexcursions at higher temperatures (G. Trager, unpublished data), whichshould make this approach technically feasible.

In summary, oral influenza vaccine based on rAd administration canelicit antibody responses to influenza in greater than 90% of subjects.While this is an early clinical stage study and several studies willneed to be completed that address issues such as interference andrepeated seasonal use, these results look encouraging for safety andimmunogenicity.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, patentapplications, websites, and database accession entries cited herein arehereby incorporated by reference in their entirety for all purposes.

REFERENCES

-   1. Kariko K, Bhuyan P, Capodici J, Weissman D. 2004. Small    interfering RNAs mediate sequence-independent gene suppression and    induce immune activation by signaling through toll-like receptor 3.    J Immunol 172:6545-6549.-   2. Boukhvalova M S, Blanco J C. 2013. The cotton rat Sigmodon    hispidus model of respiratory syncytial virus infection. Curr Top    Microbiol Immunol 372:347-358.-   3. Prince G A, Curtis S J, Yim K C, Porter D D. 2001.    Vaccine-enhanced respiratory syncytial virus disease in cotton rats    following immunization with Lot 100 or a newly prepared reference    vaccine. J Gen Virol 82:2881-2888.-   4. Liebowitz D, Lindbloom J D, Brandi J R, Garg S J, Tucker    S N. 2015. High Titer Neutralizing Antibodies to Influenza Following    Oral Tablet Immunization: A Randomized, Placebo-controlled Trial    Lancet Infect Dis 15:1041-1048.-   5. Schagen F H, Rademaker H J, Fallaux F J, Hoeben R C. 2000.    Insertion vectors for gene therapy. Gene Ther 7:271-272.-   6. Harui A, Suzuki S, Kochanek S, Mitani K. 1999. Frequency and    stability of chromosomal integration of adenovirus vectors. J Virol    73:6141-6146.-   7. Russell W C. 2000. Update on adenovirus and its vectors. J Gen    Virol 81:2573-2604.-   8. Paielli D L, Wing M S, Rogulski K R, Gilbert J D, Kolozsvary A,    Kim J H, Hughes J, Schnell M, Thompson T, Freytag S O. 2000.    Evaluation of the biodistribution, persistence, toxicity, and    potential of germ-line transmission of a replication-competent human    adenovirus following intraprostatic administration in the mouse. Mol    Ther 1:263-274.-   9. Sheets R L, Stein J, Bailer R T, Koup R A, Andrews C, Nason M, He    B, Koo E, Trotter H, Duffy C, Manetz T S, Gomez P. 2008.    Biodistribution and toxicological safety of adenovirus type 5 and    type 35 vectored vaccines against human immunodeficiency virus-1    (HIV-1), Ebola, or Marburg are similar despite differing adenovirus    serotype vector, manufacturer's construct, or gene inserts. J    Immunotoxicol 5:315-335.-   10. Zhou D, Cun A, Li Y, Xiang Z, Ertl H C. 2006. A    chimpanzee-origin adenovirus vector expressing the rabies virus    glycoprotein as an oral vaccine against inhalation infection with    rabies virus. Mol Ther 14:662-672.-   11. Scallan C D, Tingley D W, Lindbloom J D, Toomey J S, Tucker    S N. 2013. An adenovirus-based vaccine with a double-stranded RNA    adjuvant protects mice and ferrets against H5N1 avian influenza in    oral delivery models. Clin Vaccine Immunol 20:85-94.

What is claimed is:
 1. A method for eliciting an immune response to anorovirus viral protein 1 (VP1) in a human, the method comprisingadministering an immunogenic composition to the human, wherein theimmunogenic composition comprises an expression vector comprising aheterologous nucleic acid encoding a VP1 polypeptide, wherein thenucleic acid has at least 90% identity to SEQ ID NO:3 or at least 90%identity to SEQ ID NO:1.
 2. The method of claim 1, wherein the VP1polypeptide has at least 95% identity to SEQ ID NO:4 or SEQ ID NO:2. 3.The method of claim 1, wherein the nucleic acid sequence has at least98% identity to SEQ ID NO:3 or at least 98% identity to SEQ ID NO:1. 4.The method of claim 1, wherein the expression vector is a viral vector.5. The method of claim 4, wherein the viral vector is an adenoviralvector.
 6. The method of claim 5, wherein the adenoviral vector is anE1/E3-deleted replication-incompetent serotype 5 adenoviral vector. 7.The method of claim 6, wherein the vector comprises a nucleic acidsequence encoding a dsRNA adjuvant.
 8. The method of claim 7, whereinthe dsRNA adjuvant is a TLR3 agonist.
 9. The method of claim 1, whereinthe expression vector is encompassed by an enteric coating having athreshold pH of 5.8-6.8.
 10. The method of claim 9, wherein entericcoating has a threshold pH of 5.9, 6.0, or-6.1.
 11. The method of claim1, wherein the expression vector is encompassed by an enteric coatingcomprising poly(methacrylic acid-co-methyl methacrylate) 1:1.
 12. Themethod of claim 11, where in the enteric coating further comprisestriethyl citrate and talc.
 13. The method of claim 12, wherein thecomposition is in the form of a compressed tablet.
 14. An immunogeniccomposition for eliciting serum IgG and intestinal secretory IgA immuneresponses in a human, the composition comprising: (i) an E1/E3-deletedreplication-incompetent serotype 5 adenoviral vector comprising anucleic acid sequence encoding a viral protein 1 (VP1) polypeptide ofnorovirus or the fusion protein (F) of respiratory syncytial virus (RSV)and a nucleic acid sequence encoding a dsRNA adjuvant, encompassed by(ii) an enteric coating having a threshold pH of 5.8-6.8 that directsdelivery and release of the adenoviral vector to the ileum of the human.15. The immunogenic composition of claim 14, wherein the enteric coatingdisintegrates at least 75% compared to its original thickness in 110minutes at pH 5.8-6.8.
 16. The immunogenic composition of claim 14,wherein the enteric coating has a threshold pH of 5.9, 6.0, or 6.1. 17.The immunogenic composition of claim 14, wherein the enteric coatingcomprises poly(methacrylic acid-co-methyl methacrylate) 1:1.
 18. Theimmunogenic composition of claim 14, wherein the enteric coating furthercomprises triethyl citrate and talc.